production d`hydrogène par bioconversion du

Transcription

Université du Québec
Institut National de la Recherche Scientifique
Centre - Eau Terre Environnement
PRODUCTION D'HYDROGÈNE PAR BIOCONVERSION DU GLYCÉROL
BRUT ET UTILISATION DURABLE DES REJETS LIQUIDES GÉNÉRÉS
AU COURS DU PROCESSUS
Par
SAURABH JYOTI SARMA
Thèse présentée pour I 'obtention du grade de
Philosophiae doctor (Ph.D.)
en sciences de I'eau
Jury d‘évaluation
Président du jury et
examinateur externe
Dr. Laura Merlo-Sosa
Atomic Energy of Canada Limited
Examinateur externe
Prof. Safia Hamoudi
Université Laval
Examinateur interne
Prof. Jean-François Blais
INRS-ETE
Directeur de recherche
Dr. Satinder Kaur Brar
INRS-ETE
Codirecteur de recherche
Dr. Yann Le Bihan
CRIQ, Québec
© Droits réservés de Saurabh Jyoti Sarma, 2014
DÉDICACE
This thesis is dedicated in memory of my father (Late) Karmi Dev Sarma
iii
REMERCIEMENTS
I am sincerely thankful to my thesis supervisor, Dr. Satinder Kaur Brar, for giving me the
opportunity to work in this particular project. I am also greatful for her intelectual input in
developing this doctoral project and for valuable suggestion during writing the research
articles as well as thesis preparation.
I would like to extend my gratitude to Dr. Yann Le Bihan (co-supervisor), Dr. Gerardo
Buelna (collaborator of the project) of CRIQ and Dr. Mausam Verma (CO2 Solutions
inc.) for technical suggestions.
My special thank goes to the examiners, Dr. Laura Merlo-Sosa (Atomic Energy of
Canada Limited), Prof. Safia Hamoudi (Université Laval), and Prof. Jean-François Blais
(INRS-ETE); who have a great contribution in improving the overall scientific quality of
my PhD project in terms of their valuable opinions.
I appreciate the kindness of Prof. Carlos Ricardo Soccol (Brazil), Prof. Damia Barcelo
(Spain) and Prof. Kannan Pakshirajan (India) for allowing me to gather practical
experiences by spending time in their respective laboratories.
Fonds de Recherche du Québec – Nature et Technologies (FRQNT), Centre de
recherche industrielle du Québec (CRIQ), and Institut National de la Recherche
Scientifique (INRS), Centre Eau, Terre & Environnement (ETE) have been
acknowledged for providing me the scholarship to carry out this doctoral study.
I am also thankful to the laboratory staff members of INRS and the summer trainees for
their valuable assistance in laboratory. Finally, I would like to thank my family members
and friends for their support and continuous encouragement.
(Saurabh Jyoti Sarma)
v
RÉSUMÉ
Le biohydrogène est une source d'énergie renouvelable, qui a l’avantage d’être le
carburant le plus propre pour l'avenir. La combustion de l'hydrogène ne produit pas de
CO2. La seule émission du processus est de l'eau et, par conséquent, ce combustible a
un potentiel de réduction des émissions de gaz à effet de serre (GES) très élevé. De
même, l’hydrogène a une densité d'énergie massique supérieure à celle des
combustibles conventionnels tels que l'essence ou le diesel. Sur une note similaire, le
biodiesel est l'un des carburants renouvelables principalement étudiés et disponibles au
niveau commercial. Au cours de la production du biodiesel par transestérification des
lipides (par exemple, les huiles végétales, les graisses animales et les lipides des
algues), du glycérol brut (GB) est généré comme sous-produit et représente 10% du
poids du produit. En raison de la présence de nombre d'impuretés ainsi que
l'augmentation continue de la production mondiale, la valeur de marché actuelle du GB
est aussi bas que $ 0.11/kg. De plus, le coût d'élimination efficace et durable du GB
contenant du méthanol, du sel et d'autres impuretés est un défi pour les fabricants de
biodiesel. En variante, le GB peut être utilisé comme matière première (substrat) pour la
production de biohydrogène. De façon conventionnelle, l'hydrogène peut également
être produit par des techniques chimiques tels que le reformage à la vapeur du gaz
naturel, l’oxydation partielle du pétrole et du charbon par la gazéification. Contrairement
à la production biologique d'hydrogène, toutes ces techniques utilisent des
combustibles fossiles comme matière première et ne sont donc pas considérés comme
des processus durables.
Dans le cadre de ce projet de doctorat, la faisabilité technico-économique de la
production d'hydrogène par la bioconversion du GB a été évaluée. Le coût le plus élevé
de production se situe au niveau du procédé de fermentation. Néanmoins, certaines
options alternatives pour la réduction du coût du procédé ont été identifiées. Une
recherche a été effectuée afin de déterminer le potentiel maximum de production de H2
à partir du GB en l'absence de tout nutriment coûteux supplémentaire. Une production
maximale de 2023 mL-H2/L-milieu a été atteint et 10 g/L de GB a été jugée la
concentration initiale optimale. En outre, l'ajout au milieu d’une quantité de biomasse
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endogène (50 mg/L) provenant d’une fermentation antérieure et dont les ressources
sont épuisées a permis d’améliorer la production de 32,5%. De même, l'application de
suppléments moins chers, tels que les rejets liquides d’abattoir (LA), les déchets de
brasserie (BDB) et l'urée ont amélioré la production de 19±4, 27±3 et 39±4%,
respectivement. Le citrate ferrique et ses particules séchées par un atomiseur ont
également amélioré la production d'hydrogène de 17,2% à 31,7%.
La production
d'hydrogène utilisant des concentrations extrêmement faibles de GB initiale de 100
mg/L a permis de produire 22,7 mol-H2/kg GB soit une valeur de 2,75 fois plus élevées
que la valeur de 8,25 mol-H2/kg GB connu pour les systèmes de fermentation sombre.
L'effet inhibiteur des impuretés contenues dans le GB d’origine industrielle, telles que le
methanol, NaCl et le savon a été étudié. Sur la base de la vérification effectuée en
utilisant la méthodologie de réponse de surface, il a été observé que le savon est le
facteur le plus significatif (valeur p = 0,0104 ˂ 0,05) pour inhiber la production
d'hydrogène. Afin de réduire l'effet inhibiteur du savon, le GB a été traité avec du
MgSO4 pour convertir le savon en une forme inactive (écume). L'approche a permis
d’augmenter la production cumulative de H2 (34,7%) ainsi que l'utilisation du glycérol
(près de 2,5 fois). Du même coup, le traitement permet d’augmenter la teneur en Mg
(un nutriment) dans le milieu passant d’une concentration de 0,57 mg/L à 201,9 mg/L.
Pour éviter l'inhibition du processus métabolique ou décalage possible en raison de
l'accumulation d'acide organique dans le milieu, lors de la fermentation, un ajustement
rigoureux du pH est nécessaire. Comme alternative, pour la première fois, un système
de bioréacteur anaérobie à deux phases a été mis au point pour l'extraction in situ des
différents acides organiques générés lors de la fermentation.
Sur la base de
biocompatibilité, l'alcool oléique a été sélectionné en tant que phase organique du
système et une augmentation de la production d'hydrogène de 26,6 mmol/L à 29,6
mmol/L-milieu de culture a été obtenu.
En ce qui concerne les expérimentations finales, un procédé semi-continu a été utilisé
pour éviter l'inhibition du substrat, ainsi que réduire l'accumulation de sous-produits
pendant la production d'hydrogène.
Le processus a été optimisé pour produire un
rendement volumique aussi haut que 5.18 L-H2/L-milieu de culture, ce qui est nettement
viii
supérieur à celui de 2,02 à 2,68 L-H2/L-milieu précédemment rapporté pour la
bioconversion du GB.
Quant au volet visant la valorisation des rejets de fermentation l’aspect solubilisation du
phosphate a été abordé.
Certains bio-fertilisants commerciaux solubilisant le
phosphate (PSB) utilisent des acides organiques pour chélater les cations de
phosphates insolubles (comme Ca3(PO4)2) présents dans le sol pour solubiliser le
phosphate et le rendre disponible pour les plantes. Il a été observé que le liquide
organique des rejets de fermentation du GB est riche en acide organique et pourrait
servir à la solubilisation du phosphate. Des essais en laboratoire ont donc été réalisés.
Lors de son ajout au sol, une augmentation de l’assimilation du phosphore de 2,18 à
2,74 fois a été mesuré par les plantes de soya.
Ces résultats suggèrent que les
déchets liquides de fermentation du GB ont le potentiel de remplacer les PSB
disponibles dans le commerce. De même, lors de la production d'hydrogène par
fermentation, en plus de H2, divers produits chimiques industriels, y compris l'éthanol, le
1,3 propanediol et de l'acide butyrique sont produits. Si ces produits ne sont pas
récupérés, le liquide résiduel peut être utilisé comme matière première pour la
production de polyhydroxyalcanoates, des lipides, CH4, H2 et de l'électricité. Ainsi, dans
le cadre de la présente recherche, un processus de production de biohydrogène a été
évalué comme une bioraffinerie ayant le potentiel de produire du biocarburant, des
produits de la chimie fine et des biomatériaux. La stratégie pourrait être utile pour
réduire le coût global du processus en générant des revenus de sources multiples.
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ABSTRACT
Biohydrogen is the renewable energy source, which has been postulated to be the
cleanest fuel for future. Hydrogen combustion does not produce CO2; sole emission of
the process is water and hence, it has very high greenhouse gas (GHG) emission
reduction potential. Likewise, H2 has a gravimetric energy density higher than
conventional fuels such as gasoline, diesel. On a similar note, biodiesel is one of the
mostly studied and commercially available renewable fuels. During biodiesel production
by transesterification of lipids (e.g. vegetable oils, animal fats and algal lipids) crude
glycerol (CG) is generated as a byproduct; and by weight, it is around 10 % of the
product. Due to presence of number of impurities as well as continuous increase in its
global production, the present market value of CG is as low as $ 0.05/pound.
Additionally, cost effective and sustainable disposal of CG containing methanol, salt and
other impurities is a challenge for biodiesel manufacturers. Alternatively, CG can be
used as a feedstock for microbial hydrogen production. Hydrogen can also be produced
by chemical techniques such as steam reforming of natural gas; partial oxidation of
heavy oil and coal gasification. Unlike biological hydrogen production, all these
techniques use fossil fuels as the feedstock and thus are not considered as sustainable
processes.
As a part of the present investigation, techno-economic feasibility of hydrogen
production by CG bioconversion has been evaluated and high process cost was found
to be the major bottleneck. Nevertheless, certain achievable alternative options for
reduction of process cost have been identified. An investigation has been carried out to
determine maximum H2 production potential of CG in the absence of any additional
expensive supplement. A maximum production of 2022.5 mL H2/L medium has been
achieved and 10 g/L was found to be the optimum initial CG concentration. Further,
addition of spent biomass (50 mg/L) of the process into a subsequent process was
found to improve the production by 32.50%. Similarly, application of less expensive
supplements, such as slaughterhouse liquid waste (SL), brewery waste biomass (BWB)
and urea was found to improve the production by 18.81 ± 3.56, 27.30 ± 3.54 and 38.57
± 3.66 %, respectively. Ferric citrate and its nano-spray dried particles have also been
x
found to enhance hydrogen production by 17.18% to 31.71%. Hydrogen production
using extremely low initial CG concentration of 100 mg/L has been found to produce
22.7 mol-H2/kg CG; which is 2.75 folds higher than the value of 8.25 mol-H2/kg CG
known for dark fermentation.
The effect of CG impurities, such as methanol, NaCl and soap on hydrogen production
and substrate (glycerol) utilization has been studied. Based on the investigation carried
out using response surface methodology, it has been observed that soap is the most
significant factor (p value = 0.0104 ˂ 0.05) to inhibit hydrogen production. In order to
reduce the inhibitory effect of soap, CG was treated with MgSO4 to convert the soap to
its inactive form (scum). The approach was found to increase cumulative H2 production
(34.70%) as well as glycerol utilization (nearly 2.5 folds). Additionally, the treatment can
increase the Mg (a nutrient) content of the medium from 0.57 ppm to 201.92 ppm.
To avoid process termination or possible metabolic shift due to organic acid
accumulation in the medium, during fermentative hydrogen production a rigorous pH
adjustment is necessary. Alternatively, for the first time, a two phase anaerobic
bioreactor system has been developed for in situ extraction of different organic acid
byproducts. Based on biocompatibility, oleyl alcohol has been chosen as the organic
phase of the system and an increase in hydrogen production from 26.58 mmol/L to
29.61 mmol/L-medium has been achieved. Further, a semi-continuous process has
been used to avoid substrate inhibition as well as to minimize byproducts accumulation
during hydrogen production. The process was found to produce as high as 5.18 L-H2/Lmedium, which is significantly higher than 2.02 to 2.68 L-H2/L-medium previously
reported for CG bioconversion.
Organic acids produced by commercial phosphate solubilizing bio-fertilizer (PSB) can
chelate the cations of insoluble phosphates (such as Ca3 (PO4)2) present in soil to
solubilize the phosphate and make it available for plants. It has been observed that
organic acid-rich liquid waste of fermentative hydrogen production process also has
excellent phosphate solubilizing ability. Upon its addition to soil, 2.18 to 2.74 folds
increase in phosphorus uptake by soybean plants has been observed. The finding
indicates that the liquid waste has the potential to be an alternative for PSB. Likewise,
xi
during fermentative hydrogen production, in addition to H2, various industrial chemicals
including ethanol, 1,3 propanediol and butyric acid are produced. If these products are
not recovered, the liquid waste can be used as the feedstock for production of
polyhydroxyalkanoates, lipid, CH4, H2 and electricity. Thus, as a part of present
investigation, biohydrogen production process has been evaluated as a potential
biorefinery producing biofuels, fine chemicals and biomaterials. The strategy could be
useful to reduce overall cost of the process by generating revenue from multiple
sources.
xii
TABLE DES MATIÈRES
REMERCIEMENTS .............................................................................................................................. v
RÉSUMÉ ............................................................................................................................................. vii
ABSTRACT ........................................................................................................................................... x
LISTE DES TABLEAUX ................................................................................................................... xxvi
LISTE DES FIGURES .................................................................................................................... xxviii
LISTE DES ABRÉVIATIONS .......................................................................................................... xxxii
PUBLICATIONS DE CETTE THÈSE ............................................................................................ xxxiv
PUBLICATION EN DEHORS DE CETTE THÈSE....................................................................... xxxvi
CONFÉRENCES ............................................................................................................................ xxxix
CHAPITRE 1 ......................................................................................................................................... 1
SYNTHÈSE ........................................................................................................................................... 1
PARTIE I. PRODUCTION D'HYDROGENE PAR GLYCÉROL BRUT BIOCONVERSIONREVUE DE LA LITTÉRATURE ........................................................................................................... 3
1.1. Introduction ................................................................................................................................ 3
1.2. Glycérol: sous-produit de la production du biodiesel ............................................................. 6
1.2.1. Transestérification d'huiles végétales et des graisses animales:................................... 6
1.2.2. Impuretés présentes dans le glycérol brut: ...................................................................... 7
1.2.3. Problèmes environnementaux reliés aux rejets du glycérol provenant de la
production de biodiesel:................................................................................................................ 7
1.3. Glycérol: un substrat pour la production de bio-hydrogène ................................................... 7
1.3.1. Glycérol comme matière première pour la production d'hydrogène: ............................. 7
1.3.2. Métabolisme du glycérol et de la production d'hydrogène:............................................. 8
1.4. Prétraitement du glycérol brut .................................................................................................. 9
1.4.1. Importance du prétraitement du glycérol brut: ................................................................. 9
1.4.2. Différentes méthodes de prétraitements du GB: ............................................................. 9
1.5. Génie biologique pour la production d'hydrogène ............................................................... 10
1.5.1. Évaluation de différents types de réacteurs pour la production de bio-hydrogène: ... 10
1.5.2. Paramètres importants du procédé et leur niveau optimal: .......................................... 10
1.6. Microorganismes utilisés pour la bioconversion du glycerol................................................ 11
1.6.1. Microorganismes utilisés actuellement pour la bioconversion du glycérol. ................. 11
1.6.2. Génie génétique et métabolique pour l'amélioration de la production d'hydrogène:.. 12
xiii
PARTIE II. PROBLÈMES, HYPOTHÈSE, OBJECTIFS ET ORIGINALITÉ................................... 13
2.1. Problèmes ................................................................................................................................ 13
2.1.1. Coût des composantes et média supplémentaires ....................................................... 13
2.1.2. Effet inhibiteur des impuretés dans le GB (comme le savon, le méthanol, NaCl) sur la
production de H2.......................................................................................................................... 13
2.1.3. Co-production d'un grand volume de déchets liquides pendant la production de
biohydrogène ............................................................................................................................... 14
2.1.4. Effet inhibiteur du pH sur la production du H2 en fermentation sombre ...................... 14
2.1.5. Auto inhibition de la production par accumulation de H2 .............................................. 14
2.2. Hypothèse ................................................................................................................................ 15
2.3. Objectifs ................................................................................................................................... 17
2.3.1. Caractérisation du glycérol brut ...................................................................................... 18
2.3.2. Production de biohydrogène à partir de la bioconversion du GB provenant d’une
usine de production de biodiesel : évaluation technico-économique ..................................... 18
2.3.3. Évaluation de GB en tant que seule substrat pour la production d'hydrogène et
valorisation des déchets dans le processus ultérieur .............................................................. 18
2.3.4. Screening de différents déchets en tant que source de nutriments supplémentaires 18
2.3.5. Étude de l'effet des différentes impuretés du glycérol brut sur la production
d'hydrogène et de l'utilisation du glycérol par E. aerogenes utilisant du glycérol pur comme
substrat ........................................................................................................................................ 19
2.3.6. Utilisation du NaCl et MgSO4 comme stratégie de base pour l'atténuation de l'effet
inhibiteur du savon présent dans le GB .................................................................................... 19
2.3.7. Étude des effets de citrate de fer et de ses nanoparticules vaporisés (spray dryer) sur
la production d'hydrogène par bioconversion du GB ............................................................... 19
2.3.8. Développement d'un système à deux phases pour l'amélioration de la production
d'hydrogène par élimination simultanée des acides organiques du milieu ............................ 19
2.3.9. Production d'hydrogène en utilisant un bioréacteur de 7,5 L avec une libération
continue du gaz produit et la surveillance en ligne de la production d'hydrogène par un
capteur spécifique au H2 ............................................................................................................ 20
2.3.10. Évaluation de la capacité de solubilisation du phosphate des rejets liquides du
bioréacteur de production de biohydrogène et son application comme une alternative au
bioengrais utilisé pour solubiliser le phosphate ........................................................................ 20
2.4. Originalité ................................................................................................................................. 20
PARTIE III. SOMMAIRE DES DIFFÉRENTS VOLETS DE RECHERCHE EFFECTUÉS DANS
CETTE ÉTUDE ................................................................................................................................... 23
3.1. Production de biohydrogène: Principes, possibilité et défis ................................................ 23
xiv
3.1.1. Production microbienne d'hydrogène par bioconversion du glycérol brut: revue de
l’information ................................................................................................................................. 23
3.1.2. Enzymes clés: amélioration de la bioconversion du glycérol brut en biohydrogène .. 26
3.1.3. Production de biohydrogène et concept de bioraffinerie: Utilisation potentielle des
déchets liquides de la production d'hydrogène (DLPH) par fermentation.............................. 26
3.1.4. Production de bio-hydrogène par la bioconversion du glycérol brut dérivé de la
production du biodiesel: Une évaluation technico-économique.............................................. 28
3.2. Production d'hydrogène par la bioconversion du glycérol brut: Une synthèse pratique ... 28
3.2.1. Production d'hydrogène à partir du glycérol brut généré lors de la production de
biodiesel à partir de graisses animales et des déchets de restaurant par fermentation
anaérobie et ré-utilisation d’une culture carencée ................................................................... 28
3.2.2. Évaluation des différents nutriments supplémentaires pour une meilleure production
de biohydrogène par Enterobacter aerogenes NRRL B 407 à partir du glycérol brut
provenant de déchets de production du biodiesel ................................................................... 29
3.3. Effet des contaminants du glycérol brut: spéculations et faits............................................. 30
3.3.1. Étude de l'effet des différentes composantes du glycérol brut sur la production
d'hydrogène par Enterobacter aerogenes NRRL B- 407......................................................... 30
3.3.2. Atténuation de l'effet inhibiteur du savon par traitement au sel de magnésium du
glycérol brut : Une nouvelle approche pour une meilleure production de biohydrogène à
partir des déchets de l'industrie du biodiesel............................................................................ 30
3.4. Application de la nanotechnologie pour améliorer la bioconversion du glycérol brut........ 31
3.4.1. Amélioration de la production d'hydrogène par bioconversion des rejets de l’industrie
du biodiesel supplémentés avec du citrate ferrique et de ses nanoparticules générées par
un atomiseur à sec...................................................................................................................... 31
3.5. Application potentielle des rejets liquides, riches en acide organique, provenant du
procédé de production du biohydrogène pour la solubilisation du phosphate insoluble .......... 32
3.5.1. Rejets liquides de la production de biohydrogène-une alternative commercialement
intéressante en remplacement des biofertilisants solubilisant le phosphate ......................... 32
3.5.2. Solubilisation du phosphate en utilisant les rejets liquides provenant de la production
de biohydrogène (RLPH) et amélioration de l'absorption du phosphore par les plants de
soya .............................................................................................................................................. 32
3.6. Conception et optimisation de nouveaux bioprocédés pour améliorer la production
d'hydrogène en utilisant le GB ....................................................................................................... 33
3.6.1. Nouveau système anaérobie à deux phases pour la production de biohydrogène et
l'extraction in-situ des acides organiques produits................................................................... 33
3.6.2. Mise au point d’un bioprocédé en semi-continu à faible coût et suivi en continu de la
production d'hydrogène à partir du glycérol brut ...................................................................... 33
xv
References ...................................................................................................................................... 35
CHAPTER 2 ........................................................................................................................................ 41
BIOHYDROGEN PRODUCTION: PRINCIPLES, POSSIBILITY AND CHALLENGES ................ 41
PART I ................................................................................................................................................. 43
MICROBIAL HYDROGEN PRODUCTION BY BIOCONVERSION OF CRUDE GLYCEROL: A
REVIEW .............................................................................................................................................. 43
Résumé ........................................................................................................................................... 44
Abstract............................................................................................................................................ 45
Introduction ...................................................................................................................................... 46
Glycerol: by-product of biodiesel production ................................................................................ 47
Transesterification of vegetable oils and animal fats ............................................................... 47
Global biodiesel market and crude glycerol production:.......................................................... 48
The impurities present in crude glycerol: .................................................................................. 49
Environmental problems of glycerol containing biodiesel waste:............................................ 50
Different applications of crude glycerol: .................................................................................... 50
Glycerol: a substrate for bio-hydrogen production ....................................................................... 51
Different substrates presently used for bio-hydrogen: ............................................................. 51
Glycerol as a feedstock for hydrogen production:.................................................................... 52
Glycerol metabolism and hydrogen production: ....................................................................... 54
Justification of crude glycerol bioconversion to hydrogen: ...................................................... 56
Challenges to be addressed: ..................................................................................................... 57
Pretreatment of crude glycerol....................................................................................................... 59
Importance of crude glycerol pretreatment: .............................................................................. 59
Different methods of crude glycerol pretreatment: ................................................................... 60
Bioreactor systems for hydrogen production ................................................................................ 61
Different types of reactors evaluated for bio-hydrogen production:........................................ 61
Important process parameters and their optimum levels: ....................................................... 63
Microorganisms used for glycerol bioconversion ......................................................................... 64
Microorganisms presently used for glycerol bioconversion:.................................................... 64
Genetic and metabolic engineering for improved hydrogen production:................................ 67
Conclusion and future perspectives .............................................................................................. 68
Abbreviations................................................................................................................................... 69
xvi
Acknowledgments........................................................................................................................... 69
References ...................................................................................................................................... 71
PART-II .............................................................................................................................................. 105
KEY ENZYMES IN VALUE-ADDITION OF CRUDE GLYCEROL TO BIOHYDROGEN ........... 105
Résumé ......................................................................................................................................... 106
Abstract.......................................................................................................................................... 107
Introduction .................................................................................................................................... 108
Important Metabolic Pathways..................................................................................................... 109
Glycerol Bioconversion Pathways ........................................................................................... 109
Hydrogen Production Pathways .............................................................................................. 110
Hydrogenases ............................................................................................................................... 113
[NiFe] -hydrogenases ............................................................................................................... 114
[FeFe]-hydrogenases................................................................................................................ 116
Nitrogenases ................................................................................................................................. 117
Recent Metabolic/Enzyme Engineering Strategies for Improved CG Bioconversion and H2
Production ..................................................................................................................................... 119
Conclusions ................................................................................................................................... 121
Acknowledgments......................................................................................................................... 122
References .................................................................................................................................... 123
PART-III ............................................................................................................................................. 141
HYDROGEN BIOREFINERY: POTENTIAL UTILIZATION OF THE LIQUID WASTE FROM
FERMENTATIVE HYDROGEN PRODUCTION ............................................................................ 141
Résumé ......................................................................................................................................... 142
Abstract.......................................................................................................................................... 143
Introduction .................................................................................................................................... 144
Characteristics of the liquid waste from biohydrogen production process............................... 145
Ethanol recovery from HPLW ...................................................................................................... 147
1,3-propanediol recovery from HPLW ......................................................................................... 149
Butyric acid recovery from HPLW ............................................................................................... 150
Methane production from HPLW ................................................................................................. 152
Bioplastic (PHA) production from HPLW .................................................................................... 153
Potential application of HPLW as phosphate solubilizer ........................................................... 155
xvii
Other products from HPLW .......................................................................................................... 156
Concluding statements ................................................................................................................. 158
References .................................................................................................................................... 161
PART-IV ............................................................................................................................................ 189
BIO-HYDROGEN PRODUCTION BY BIODIESEL DERIVED CRUDE GLYCEROL
BIOCONVERSION: A TECHNO-ECONOMIC EVALUATION ...................................................... 189
Résumé ......................................................................................................................................... 190
Abstract.......................................................................................................................................... 191
Introduction .................................................................................................................................... 192
Biodiesel and CG production in NA............................................................................................. 193
CG bioconversion process overview ........................................................................................... 195
Inoculum preparation ................................................................................................................ 196
CG pre-treatment ...................................................................................................................... 196
Media preparation ..................................................................................................................... 197
Anaerobic dark fermentation .................................................................................................... 197
Photo fermentation.................................................................................................................... 198
Treatment of waste and biogas production............................................................................. 198
Building construction, equipment & labour ............................................................................. 199
Hydrogen yield .......................................................................................................................... 199
A cost benefit analysis of CG bioconversion process................................................................ 200
Estimation of environmental benefit of CG bioconversion process .......................................... 202
GHG emission reduction .......................................................................................................... 202
An outlook on other environmental benefits ........................................................................... 203
Conclusions ................................................................................................................................... 204
Abbreviations................................................................................................................................. 205
References .................................................................................................................................... 206
CHAPTER 3 ...................................................................................................................................... 221
HYDROGEN PRODUCTION BY CRUDE GLYCEROL BIOCONVERSION: A PRACTICAL
SUMMARY ........................................................................................................................................ 221
PART I ............................................................................................................................................... 223
xviii
HYDROGEN PRODUCTION FROM MEAT PROCESSING AND RESTAURANT WASTE
DERIVED CRUDE GLYCEROL BY ANAEROBIC FERMENTATION AND UTILIZATION OF
THE SPENT BROTH........................................................................................................................ 223
Résumé ......................................................................................................................................... 224
Abstract.......................................................................................................................................... 225
Introduction .................................................................................................................................... 226
Materials and methods ................................................................................................................. 227
Microorganism and inoculum development ............................................................................ 227
Chemicals and reagents........................................................................................................... 228
Characterization of CG ............................................................................................................. 228
Investigation of the effect of CG concentration on hydrogen production ............................. 229
Characterization of spent media fraction ................................................................................ 230
Utilization of spent media fraction in subsequent process for H2 production ....................... 230
Utilization of active biomass from spent media ...................................................................... 230
Utilization of spent biomass as a supplementary nutrient ..................................................... 231
Analysis...................................................................................................................................... 231
Glycerol analysis ....................................................................................................................... 231
Hydrogen analysis .................................................................................................................... 232
Results and discussion................................................................................................................. 232
Crude glycerol characterization ............................................................................................... 232
Hydrogen production by CG bioconversion ............................................................................ 233
Utilization of spent broth of H2 production process ................................................................ 236
Conclusions ................................................................................................................................... 238
References .................................................................................................................................... 240
PART II .............................................................................................................................................. 253
EVALUATION OF DIFFERENT SUPPLEMENTARY NUTRIENTS FOR ENHANCED
BIOHYDROGEN PRODUCTION BY ENTEROBACTER AEROGENES NRRL B 407 USING
WASTE DERIVED CRUDE GLYCEROL ....................................................................................... 253
Résumé ......................................................................................................................................... 254
Abstract.......................................................................................................................................... 255
Introduction .................................................................................................................................... 256
Materials and Methods ................................................................................................................. 257
xix
Microorganism and inoculum development ............................................................................ 257
Chemicals and other materials ................................................................................................ 258
Characterization of crude glycerol ........................................................................................... 258
Hydrogen production experiments........................................................................................... 259
Evaluation of organic waste materials as supplementary nutrient source for CG
bioconversion ............................................................................................................................ 260
Evaluation of urea as supplementary nitrogen source for CG bioconversion...................... 261
Analytical methods .................................................................................................................... 261
Glycerol ...................................................................................................................................... 261
Evaluation of different waste materials as nutrient supplements.......................................... 263
Effect of urea on H2 production using CG ............................................................................... 266
Conclusions ................................................................................................................................... 267
References .................................................................................................................................... 269
CHAPTER 4 ...................................................................................................................................... 283
EFFECT OF CRUDE GLYCEROL IMPURITIES: SPECULATIONS AND FACTS..................... 283
PART I ............................................................................................................................................... 285
INVESTIGATION OF THE EFFECT OF DIFFERENT CRUDE GLYCEROL COMPONENTS ON
HYDROGEN PRODUCTION BY ENTEROBACTER AEROGENES NRRL B-407 .................... 285
Résumé ......................................................................................................................................... 286
Abstract.......................................................................................................................................... 287
Introduction .................................................................................................................................... 288
Chemicals .................................................................................................................................. 289
Microorganism and inoculum preparation ............................................................................... 289
Saponification of crude glycerol and separation of soap ....................................................... 290
Experimental design and hydrogen production experiments ................................................ 290
Analytical techniques ................................................................................................................ 292
xx
Effect of the impurities on cumulative H2 production ............................................................. 293
Effect of the impurities on glycerol utilization.......................................................................... 294
Conclusions ................................................................................................................................... 297
References .................................................................................................................................... 298
PART II .............................................................................................................................................. 315
MITIGATION OF THE INHIBITORY EFFECT OF SOAP BY MAGNESIUM SALT TREATMENT
OF CRUDE GLYCEROL- A NOVEL APPROACH FOR ENHANCED BIOHYDROGEN
PRODUCTION FROM THE BIODIESEL INDUSTRY WASTE .................................................... 315
Résumé ......................................................................................................................................... 316
Abstract.......................................................................................................................................... 317
Introduction .................................................................................................................................... 318
Chemicals and reagents........................................................................................................... 319
Microorganism and culture conditions..................................................................................... 319
CG pretreatment using NaCl.................................................................................................... 320
Bio-hydrogen production using NaCl treated CG ................................................................... 320
Bio-hydrogen production using MgSO4 treated CG ............................................................... 321
Analytical methods .................................................................................................................... 321
Determination of soap concentration in CG samples ............................................................ 321
NaCl pretreatment and bio-hydrogen production using treated CG ..................................... 322
Biohydrogen production using MgSO4 treated CG ................................................................ 324
Conclusions ................................................................................................................................... 326
References .................................................................................................................................... 327
CHAPTER 5 ...................................................................................................................................... 335
xxi
APPLICATION OF NANOTECHNOLOGY FOR IMPROVED CRUDE GLYCEROL
BIOCONVERSION ........................................................................................................................... 335
ENRICHED HYDROGEN PRODUCTION BY BIOCONVERSION OF BIODIESEL WASTE
SUPPLEMENTED WITH FERRIC CITRATE AND ITS NANO-SPRAY DRIED PARTICLES ... 337
Résumé ......................................................................................................................................... 338
Abstract.......................................................................................................................................... 339
Introduction .................................................................................................................................... 340
Materials and Methods ................................................................................................................. 341
Preparation and characterization of different nano-spray dried particles............................. 342
Screening of different nano-spray dried particles for biohydrogen production .................... 343
Investigation of the effect of Fe-citrate particle preparation method on hydrogen production
.................................................................................................................................................... 343
Investigation of the effect of CG concentration on H2 production using media supplemented
with ferric citrate particles ......................................................................................................... 343
Results and Discussion ................................................................................................................ 344
Characterization of nano-spray dried particles ....................................................................... 344
Screening of different nano-spray dried particles for biohydrogen production .................... 345
Effect of Fe-citrate particle preparation method on hydrogen production ............................ 346
Effect of CG concentration on H2 production using media supplemented with nano-spray
dried ferric citrate particles ....................................................................................................... 347
Conclusions ................................................................................................................................... 349
References .................................................................................................................................... 350
CHAPTER 6 ...................................................................................................................................... 361
POTENTIAL APPLICATION OF ORGANIC ACID RICH LIQUID WASTE FROM HYDROGEN
PRODUCTION PROCESS FOR PHOSPHATE SOLUBILIZATION ............................................ 361
PART I ............................................................................................................................................... 363
LIQUID WASTE FROM BIO-HYDROGEN PRODUCTION - A COMMERCIALLY ATTRACTIVE
ALTERNATIVE FOR PHOSPHATE SOLUBILIZING BIO-FERTILIZER...................................... 363
Résumé ......................................................................................................................................... 364
Abstract.......................................................................................................................................... 365
Introduction .................................................................................................................................... 366
xxii
Hydrogen production ................................................................................................................ 367
Phosphate solubilization study ................................................................................................ 367
Analysis...................................................................................................................................... 368
Conclusions ................................................................................................................................... 370
References .................................................................................................................................... 372
PART II .............................................................................................................................................. 377
NOVEL ENVIRONMENTAL AND AGRICULTURAL USES OF BIOHYDROGEN PRODUCTION
WASTEWATER-A STUDY USING SOYBEAN PLANTS .............................................................. 377
Résumé ......................................................................................................................................... 378
Abstract.......................................................................................................................................... 379
Introduction .................................................................................................................................... 380
Biohydrogen production from crude glycerol by using a 7.5 L bioreactor ............................ 382
Investigation of phosphorus uptake by soybean plant in presence of HPLW ...................... 382
Analytical techniques ................................................................................................................ 383
Characterization of HPLW ........................................................................................................ 383
Phosphorus analysis................................................................................................................. 383
Measurement of biomass production ...................................................................................... 384
Determination of soil pH ........................................................................................................... 384
Results ........................................................................................................................................... 384
Biohydrogen production from crude glycerol .......................................................................... 384
Investigation of phosphorus uptake by soybean plant in presence of HPLW ...................... 385
Discussion ..................................................................................................................................... 385
Biohydrogen production from crude glycerol .......................................................................... 385
Phosphorus uptake by soybean plant in presence of HPLW ................................................ 386
References .................................................................................................................................... 390
xxiii
CHAPTER 7 ...................................................................................................................................... 399
DESIGNING AND OPTIMIZATION OF NOVEL BIOPROCESSES FOR IMPROVE HYDROGEN
PRODUCTION USING CG .............................................................................................................. 399
PART I ............................................................................................................................................... 401
A NOVEL ANAEROBIC TWO PHASE SYSTEM FOR BIOHYDROGEN PRODUCTION AND IN
SITU EXTRACTION ORGANIC ACID BYPRODUCTS ................................................................ 401
Résumé ......................................................................................................................................... 402
Abstract.......................................................................................................................................... 403
Introduction .................................................................................................................................... 404
Screening of different organic solvents for two phase system .............................................. 405
Hydrogen production with selected organic solvents............................................................. 406
Investigation of the effect of oleyl alcohol and CG concentration on hydrogen production 407
Analysis...................................................................................................................................... 407
Analysis of fermentation end products .................................................................................... 407
Screening of different organic solvents for two phase system .............................................. 408
Hydrogen production with selected organic solvents............................................................. 409
Investigation of the effect of oleyl alcohol volume and CG concentration on hydrogen
production using proposed two phase system ....................................................................... 409
Conclusions ................................................................................................................................... 411
References .................................................................................................................................... 412
PART II .............................................................................................................................................. 421
LOW COST SEMI-CONTINUOUS BIOPROCESS AND ONLINE MONITORING OF
HYDROGEN PRODUCTION FROM CRUDE GLYCEROL .......................................................... 421
Résumé ......................................................................................................................................... 422
Abstract.......................................................................................................................................... 423
Introduction .................................................................................................................................... 424
xxiv
Crude glycerol as feedstock ..................................................................................................... 425
Hydrogen production using a 7.5 L bioreactor ....................................................................... 426
Analysis...................................................................................................................................... 427
Analysis of different fermentation end products ..................................................................... 428
Results and Discussion ................................................................................................................ 429
Hydrogen production using a 7.5 L bioreactor ....................................................................... 429
Conclusions ................................................................................................................................... 433
References .................................................................................................................................... 435
CHAPITRE 8 ..................................................................................................................................... 445
CONCLUSIONS ET RECOMMANDATIONS ................................................................................. 445
Conclusions ................................................................................................................................... 447
Recommandations pour l'avenir .................................................................................................. 450
ANNEXES ......................................................................................................................................... 451
ANNEXE I.......................................................................................................................................... 452
ANNEXE II......................................................................................................................................... 454
ANNEXE III........................................................................................................................................ 456
ANNEXE IV ....................................................................................................................................... 461
ANNEXE V ........................................................................................................................................ 463
ANNEXE VI ....................................................................................................................................... 466
ANNEXE VII ...................................................................................................................................... 468
ANNEXE VIII ..................................................................................................................................... 470
ANNEXE IX ....................................................................................................................................... 472
xxv
LISTE DES TABLEAUX
Table 2.1. 1: Different valuable products obtained from crude glycerol generated during biodiesel production ................................................................................................................................. 82
Table 2.1. 2: Maximum hydrogen production by different inocula using different non-glycerol
feedstock (Hak-Joo Kim et al., 2004) ................................................................................................ 85
Table 2.1. 3: Maximum hydrogen production by different inocula using pure and crude glycerol
as feedstock ........................................................................................................................................ 87
Table 2.1. 4: Different bioreactor systems used for H2 production using glycerol and other
feedstock ............................................................................................................................................. 89
Table 2.1. 5: Microorganisms used for bioconversion of pure and crude glycerol to H2 and other
products ............................................................................................................................................... 94
Table 2.2. 1: Recent genetic/ enzyme engineering strategies for improved substrate utilization
............................................................................................................................................................ 134
Table 2.3. 1: Summary of different reports on simultaneous hydrogen and ethanol production174
Table 2.3. 2: Ethanol production by microbial conversion of different organic substrates ......... 177
Table 2.3. 3: Summary of different reports on simultaneous hydrogen and 1, 3-propanediol
production .......................................................................................................................................... 178
Table 2.3. 4: Summary of different reports on simultaneous hydrogen and butyric acid
production .......................................................................................................................................... 180
Table 2.3. 5: Bio-methane production using dark fermentative hydrogen production liquid waste
............................................................................................................................................................ 183
Table 2.4. 1: Feedstock availability and annual biodiesel and CG production in North America
............................................................................................................................................................ 210
Table 2.4. 2: Cost distribution of crude glycerol (1 Kg) bioconversion and hydrogen production
using a two-stage process ............................................................................................................... 211
Table 2.4. 3: Hydrogen production by different fermentation techniques using crude glycerol as
a cheap substrate ............................................................................................................................. 213
Table 2.4. 4: Estimation of environmental benefit from bioconversion of 1 Kg crude glycerol
using a two-stage process ............................................................................................................... 215
Table 2.4. 5: Table summarizing possible benefits of the crude glycerol bioconversion process
considered in the present study ...................................................................................................... 217
xxvi
Table 3.1. 1: Summary of different recent investigations on CG bioconversion and H2 production
............................................................................................................................................................ 243
Table 3.1. 2: Characterization of crude glycerol and spent broth of H2 production process ...... 245
Table 3.2. 1: The summary of different reports available on H2 production by CG bioconversion
............................................................................................................................................................ 273
Table 4.1. 1: Distribution of different impurities of biodiesel derived crude glycerol ................... 301
Table 4.1. 2: Central composite design ranges of the three factors considered for present
investigation. ..................................................................................................................................... 302
Table 4.1. 3: Experimental design and the responses obtained for 20 different experiments
carried out during the present study ............................................................................................... 303
Table 4.1. 4: ANOVA for response surface linear model used for response 1 (hydrogen
production) ........................................................................................................................................ 305
Table 5.1. 1: Summary of different reports on hydrogen production by crude glycerol
bioconversion .................................................................................................................................... 353
Table 6.1. 1: Possible benefit of proposed technology compared to conventional PSB ............ 374
Table 6.2. 1: Soil composition of different pots used to grow soybean plants (HPLW, hydrogen
production liquid waste; TCP, Tricalcium phosphate). .................................................................. 393
Table 7.2. 1: Summary of the present investigation ...................................................................... 437
Table 7.2. 2: Summary of different studies on hydrogen production by CG bioconversion ....... 438
Table 7.2. 3: Cost analysis of the semi-continuous process considered in the present study .. 440
xxvii
LISTE DES FIGURES
Figure 1. 1: Présentation de la thèse. ............................................................................................... 25
Figure 2.1.1: Schematic representation of a general biodiesel production process using
transesterification of vegetable oil and fats (adapted from (Hak-Joo Kim et al., 2004, Muniyappa
et al., 1996, Schuchardt et al., 1998))............................................................................................... 97
Figure 2.1.2: Equation showing transesterification of large branched triglyceride molecule to
biodiesel and glycerol (Muniyappa et al., 1996)............................................................................... 98
Figure 2.1.3: Global biodiesel production share of different geographic regions of the world till
2009 ..................................................................................................................................................... 99
Figure 2.1.4: Schematic representation of glycerol metabolism during anaerobic fermentation
(Adapted from (Biebl et al., 1999, Drożdżyńska et al., 2011)) ...................................................... 100
Figure 2.1.5: Stoichiometric equations showing hydrogen yield during glycerol bioconversion 101
Figure 2.1.6: A two-chambered microbial electrolysis cell used for H2 production by
bioconversion of glycerol (Escapa et al., 2009, Hongqiang Hu, 2009, Hong Liu et al., 2005,
Logan et al., 2008)............................................................................................................................ 102
Figure 2.1.7: Anaerobic metabolism of glucose and pyruvate and hydrogen production (Adapted
from (Chin et al., 2003, Mathews et al., 2009) ............................................................................... 103
Figure 2.2. 1: Schematic representation of the glycerol bioconversion pathways. Adapted from
(Biebl et al., 1999, Drożdżyńska et al., 2011, Sarma et al., 2012). .............................................. 136
Figure 2.2. 2: Simplified representation of PFOR pathway for hydrogen production. (a-d) shows
4 different mechanisms involved in hydrogen evolution where, PFOR= Pyruvate: ferredoxin
oxido reductase, Fd ox/red= Oxidized or reduced ferredoxin, HYD= (hydrogenase), NADH-HYD=
(NADH-dependent [FeFe] hydrogenase), NADH-Fd-HYD= (NADH- ferredoxin-dependent
hydrogenase) and NFOR= (NADH-ferredoxin oxidoreductase) (adapted from (Chen et al., 2006,
Hallenbeck et al., 2012a, Yu et al., 2011)). .................................................................................... 137
Figure 2.2. 3: Simplified representation of PFL pathway for hydrogen production (adapted from
(Hallenbeck et al., 2012a))............................................................................................................... 138
Figure 2.2. 4: Simplified representation of pentose phosphate pathway for hydrogen production
(adapted from (Y.M. Kim et al., 2011b, Veit et al., 2008))............................................................. 139
Figure 2.3. 1: Possible applications of the liquid waste from dark fermentative hydrogen
production.......................................................................................................................................... 185
Figure 2.3. 2: Reported concentration levels of different metabolites present in HPLW and their
market price in US dollars (Han et al., 2010, Lalaurette et al., 2009, Selembo et al., 2009). .... 186
xxviii
Figure 2.3. 3: Free energy of formation (25º C) of different metabolites produced during
biohydrogen production.................................................................................................................... 187
Figure 2.4. 1: Schematic representation of the process considered for the present estimation 218
Figure 2.4. 2: Cost distribution of the crude glycerol bioconversion process .............................. 219
Figure 2.4. 3: Electricity requirement of a two-stage fermentation process for CG bioconversion
and H2 production ............................................................................................................................. 220
Figure 3.1. 1: Batch H2 production (solid lines) and glycerol utilization (dotted lines) profiles
obtained for different initial CG concentrations .............................................................................. 247
Figure 3.1. 2: pH profiles obtained from the batch experiments conducted for different (initial)
CG concentrations ............................................................................................................................ 248
Figure 3.1. 3: H2 production and pH profile obtained for 10 g/L CG showing the production of
nearly 75% of H2 at pH 3.8 .............................................................................................................. 249
Figure 3.1. 4: Batch H2 production profiles obtained by addition of different spent media fractions
............................................................................................................................................................ 250
Figure 3.1. 5: Batch H2 production profiles obtained by addition of different amount of active
biomass from previous fermentation ............................................................................................... 251
Figure 3.1. 6: Batch H2 production profiles obtained by addition of different concentrations of
spent biomass (autoclaved) from previous fermentation .............................................................. 252
Figure 3.2. 1: H2 production profiles obtained for different concentrations SL (a), AS (b), SS (c),
BWL (d), BWB (e) and SIW (f) supplemented to 10 g/L CG ......................................................... 280
Figure 3.2. 2: Maximum cumulative H2 production (mmol/L), glycerol utilization (%) and broth pH
recorded at the end of fermentation for different material supplemented to 10 g/L CG. The
control indicates the results obtained for 10 g/L CG without any additive. .................................. 281
Figure 3.2. 3: H2 production profiles obtained for different concentrations urea supplemented to
10 g/L CG .......................................................................................................................................... 282
Figure 4.1. 1: Schematic representation of soap separation from crude glycerol sample ......... 307
Figure 4.1. 2: Response surface graph showing the effect of methanol on hydrogen production
............................................................................................................................................................ 308
Figure 4.1. 3: Response surface graph showing the effect of NaCl on hydrogen production.... 309
Figure 4.1. 4: Response surface graph showing the effect of soap on hydrogen production .... 310
Figure 4.1. 5: Response surface graphs showing the effect of (a) methanol and NaCl; (b) NaCl
and soap and (c) soap and methanol on glycerol utilization ......................................................... 313
xxix
Figure 4.1. 6: Hydrogen production profile obtained for the control experiment carried out in the
absence of any impurity ................................................................................................................... 314
Figure 4.2. 1: Cumulative H2 production (after 120h) obtained using 10 g/L CG (treated with
different concentration of NaCl). Initial NaCl concentration in the medium of each run of
experiment was also shown............................................................................................................. 330
Figure 4.2. 2: Cumulative H2 production obtained using 200 g/L NaCl treated CG diluted to an
initial NaCl concentration of 0.5 g/L (as from Figure 1, 0.5 g/L NaCl was found to be optimum).
Control represents result obtained for equivalent amount of untreated CG ................................ 331
Figure 4.2. 3: H2 production (a) and glycerol utilization (b) profiles obtained for 20 g/L CG
treated with different concentrations of MgSO4. Control represents result obtained for untreated
CG ...................................................................................................................................................... 333
Figure 5.1. 1: SEM images (at different magnification) of ferric citrate particles prepared using
hot water ............................................................................................................................................ 355
Figure 5.1. 2: Zeta potential distribution of ferric citrate particles prepared using hot water ..... 356
Figure 5.1. 3: Cumulative hydrogen production obtained after 48 h of incubation. 50 mg/L of
respective nanospray dried particles were added to 12 g/L of CG .............................................. 357
Figure 5.1. 4: Cumulative hydrogen production obtained after 48 h of incubation. Different
concentrations of two different types of ferric citrate particles were added to 12 g/L of CG ...... 358
ferric citrated particles (prepared with hot water) were added to different concentrations of CG.
Projected hydrogen yields by bioconversion of 1 kg of CG have also been shown for each initial
CG concentration .............................................................................................................................. 359
Figure 5.1. 6: Cumulative hydrogen production obtained after 48 h of incubation. Control is only
CG (100 mg/L) .................................................................................................................................. 360
Figure 6.1. 1: A schematic representation of the proposed concept............................................ 375
Figure 6.1. 2: Phosphate solubilization achieved using different concentrations of bio-hydrogen
production liquid waste. The control represents soluble phosphate concentration obtained using
only water as the solubilizing agent ................................................................................................ 376
Figure 6.2. 1: A schematic representation of the approach considered for the present
investigation. E-1 (Lipid), E-2 (Methanol), E-3 (NaOH/catalyst), E-4 (Transesterification), E-5
(Biodiesel processing), E-6 (Pump), E-7 (Biodiesel distribution), E-8 (Crude glycerol
fermentation), E-9 (Organic acid rich liquid waste), E-10 (Agricultural field), E-11 (Gas
xxx
separator), E-12 (Gas holder-CO2), E-13 (Gas holder-H2), E-14 (Compressor), E-15 (H2cylinders); P (Pipeline); V (Valve). .................................................................................................. 394
Figure 6.2. 2: (a) Values of different process parameters recorded online during hydrogen
production using a 7.5L fermenter. (b) Gas chromatogram of hydrogen production liquid waste
(HPLW) showing the presence of acetic acid and butyric acid as major byproducts. ................ 395
Figure 6.2. 3: Phosphorus uptake by soybean plants cultivated in the presence of different
amounts of hydrogen production liquid waste (HPLW) and tricalcium phosphate (TCP). ......... 396
Figure 6.2. 4: Effect of hydrogen production liquid waste (HPLW) and tricalcium phosphate
(TCP) treatment of soil on soybean plant biomass production and seed germination. .............. 397
Figure 7.1. 1: Schematic representation of the proposed method ............................................... 414
Figure 7.1. 2: Screening of organic phase based on biocompatibility & final media pH............. 415
Figure 7.1. 3: Screening of organic phases based on hydrogen production ............................... 416
Figure 7.1. 4: Investigation of the effect of oleyl alcohol: aqueous phase ratio on hydrogen
production .......................................................................................................................................... 417
Figure 7.1. 5: Fermentation end products detected in the aqueous phase of the reactor. (a)
Single phase control, (b) two phase process containing oleyl alcohol at an organic: aqueous
phase ratio of 1: 50. .......................................................................................................................... 418
Figure 7.1. 6: Investigation of the effect of high CG concentration on hydrogen production using
novel two phase system ................................................................................................................... 419
Figure 7.2. 1: Gas chromatogram of CG showing the presence of acetone, butanol, ethanol and
butyric acid as well as different unidentified compounds. ............................................................. 441
Figure 7.2. 2: Online data collected from the first batch of fermentation carried out using a feed
CG concentration of 60 g/L. Hydrogen accumulated in the headspace of the reactor was
periodically removed by bubbling with nitrogen gas. ..................................................................... 442
Figure 7.2. 3: Online data collected from the second batch of fermentation carried out using a
feed CG concentration of 120 g/L. Hydrogen accumulated in the headspace of the reactor was
periodically removed by bubbling with nitrogen gas. ..................................................................... 443
Figure 7.2. 4: Gas chromatogram of fermented broth after 120 hours of fermentative hydrogen
production. Ethanol has been identified as the major byproduct of the process. (a) First batch of
fermentation carried out using a feed CG concentration of 60 g/L, (b) second batch of
fermentation carried out using a feed CG concentration of 120 g/L. ........................................... 444
xxxi
LISTE DES ABRÉVIATIONS
AS
apple pomace sludge
BLW
brewery liquid waste
CAGR
compound annual growth rate
CFR
code of federal regulations
CG
crude glycerol
COD
chemical oxidation demand
CSTR
continuous stirred tank reactor
DHA
docosahexaenoic acid
EPA
Environmental protection agency, US
EU
European Union
Fd
ferredoxin
GDHt
glycerol dehydratase
GHG
greenhouse gas
GWh
gigawatt hour
HAP
hydroxyapatite
ICP-AES
inductively coupled plasma atomic emission spectroscopy
kWh
kilowatt hour
MFC
microbial fuel cell
mM
millimolar
MMt
million metric tonnes
MONG
matter organic non-glycerol
MSDS
material safety data sheet
xxxii
NA
North America
NAD+
Nicotinamide adenine dinucleotide
NAPL
non-aqueous phase liquid
PD
1,3-propanediol
PDOR
1, 3-propanediol oxidoreductase
POTW
publicly owned treatment works
PSB
phosphate solubilizing bio-fertilizer
RFS2
revised renewable fuel standard 2
RSM
response surface methodology
SIW
starch industry wastewater
SL
slaughterhouse liquid wastes
SS
secondary sludge
TOC
total organic carbon
xxxiii
PUBLICATIONS DE CETTE THÈSE
1. Saurabh Jyoti Sarma, S. K. Brar, E. B. Sydney, Y. L. Bihan, G. Buelna, C. R. Soccol.
Microbial hydrogen production by bioconversion of crude glycerol: a review. International
Journal of Hydrogen Energy, (2012), 37 (8): 6473–6490.
2. Saurabh Jyoti Sarma, Gurpreet Singh Dhillon, Satinder Kaur Brar, Yann Le Bihan, Gerardo
Buelna (2013). Key enzymes in value-addition of crude glycerol to biohydrogen. Enzymes in
Value-Addition of Wastes. Nova Science Publishers, Inc. (published).
3. Saurabh Jyoti Sarma, Pachapur V, Brar SK, Le Bihan Y, Buelna G. Hydrogen biorefinery:
Potential utilization of the liquid waste from fermentative hydrogen production. Renewable &
Sustainable Energy Reviews (Submitted).
4. Saurabh Jyoti Sarma, S. K. Brar, Y. L. Bihan, G. Buelna, Bio-hydrogen production by
biodiesel-derived crude glycerol bioconversion: a techno-economic evaluation. Bioprocess
and Biosystems Engineering (2012, DOI 10.1007/s00449-012-0755-8).
5. Saurabh Jyoti Sarma, Brar SK, Le Bihan Y, Buelna G, Rabeb L, Soccol CR (2013).
Hydrogen production from meat processing and restaurant waste derived crude glycerol by
anaerobic fermentation and utilization of the spent broth. Journal of Chemical Technology &
Biotechnology: DOI: 10.1002/jctb.4099.
6. Saurabh Jyoti Sarma, Brar SK, Le Bihan Y, Buelna G, Rabeb L, Soccol CR, et al.
Evaluation of different supplementary nutrients for enhanced biohydrogen production by
Enterobacter aerogenes NRRL B 407 using waste derived crude glycerol. International
Journal of Hydrogen Energy (2013), 38 (5): 2191–2198.
7. Saurabh Jyoti Sarma, Gurpreet Singh Dhillon, Satinder Kaur Brar, Yann Le Bihan, Gerardo
Buelna, Mausam Verma. Investigation of the effect of different crude glycerol components
on hydrogen production by Enterobacter aerogenes NRRL B-407. Renewable Energy
(2013) 60: 566–571.
8. Saurabh Jyoti Sarma, Brar SK, Le Bihan Y, Buelna G, Soccol CR. Mitigation of the
inhibitory effect of soap by magnesium salt treatment of crude glycerol- a novel approach for
enhanced biohydrogen production from the biodiesel industry waste. Bioresource
Technology, (2014) 151:49-53.
9. Saurabh Jyoti Sarma, Brar SK, Le Bihan Y, Buelna G. Improved hydrogen production by
bioconversion of biodiesel waste supplemented with ferric citrate and its nano-spray dried
particles. RSC Advances (2014) 4: 49588-49594.
xxxiv
10. Saurabh Jyoti Sarma, Satinder Kaur Brar, Yann Le Bihan, Gerardo Buelna (2013). Liquid
waste from bio-hydrogen production - a commercially attractive alternative for phosphate
solubilizing bio-fertilizer. International Journal of Hydrogen Energy (2013) 38 (21): 8704–
8707.
11. Saurabh Jyoti Sarma, Brar SK, Le Bihan Y, Buelna G. Novel environmental and
agricultural uses of biohydrogen production wastewater-A study using soybean plants.
Journal of environmental management (Submitted).
12. Saurabh Jyoti Sarma, Brar SK, Le Bihan Y, Buelna G. A novel anaerobic two phase
system for biohydrogen production and in situ extraction organic acid byproducts.
Bioprocess and Biosystems Engineering (Submitted).
13. Saurabh Jyoti Sarma, Brar SK, Le Bihan Y, Buelna G, Verma M. Low cost semi-continuous
bioprocess
and
online
monitoring
of
hydrogen
Environmental Science and Technology (submitted).
xxxv
production
from
crude
glycerol.
PUBLICATION EN DEHORS DE CETTE THÈSE
1. Saurabh Jyoti Sarma, Das RK, Brar SK, Le Bihan Y, Buelna G, Verma M, Soccol CR.
Application of magnesium sulfate and its nanoparticle for enhanced lipid production by
mixotrophic
cultivation
of
algae
using
biodiesel
waste.
Energy,
doi:10.1016/j.energy.2014.04.112.
2. Saurabh Jyoti Sarma, Indrani Bhattacharya, Satinder Kaur Brar, Rajeshwar Dayal Tyag
and Rao Y. Surampalli. Carbon nanotube- bioaccumulation and recent advances in
environmental monitoring. Critical Reviews in Environmental Science and Technology,
DOI:10.1080/10643389.2014.924177.
3. Saurabh Jyoti Sarma, Mausam Verma, Satinder Kaur Brar (2013). Industrial fermentation
for the production of alcoholic beverages. Fermentation Processes Engineering in the Food
Industry, Taylor and Francis Group, LLC (published).
4. Saurabh Jyoti Sarma, GS Dhillon, K Hegde and SK Brar (2013). Utilization of agroindustrial
waste
for
the
production
of
aroma
compounds
and
fragrances,
in
Biotransformation of agro-industrial wastes: high value biochemical; Springer, US
(published).
5. Saurabh Jyoti Sarma, Ratul Kumar Das, Satinder Kaur Brar and Mausam Verma (2013).
Fundamentals related to nanomaterials, chemical interactions/charactrization and their
linkage with fate and transportat and behavior of NMs in the environment. In Nanomaterials
in the Environment, publisher: American Society of Civil Engineers (ASCE), (in press).
6. Saurabh Jyoti Sarma, Ratul Kumar Das and Satinder Kaur Brar (2013) Singlet oxygen
production by organic matter and disintegration of pathogens and pollutants during
wastewater treatment - how important? Hydrology:Current Research. 4:e109. doi:
10.4172/2157-7587.1000e109.
7. Eduardo Bittencourt Sydney, Christian Larroche ,Alessandra Cristine Novak, Regis Nouaille,
Saurabh Jyoti Sarma, Satinder Kaur Brar, Vanete Thomaz Soccol, Carlos Ricardo Soccol.
Economic process to produce biohydrogen and volatile fatty acids by a mixed culture using
vinasse from sugarcane ethanol industry as nutrient source. Bioresource technology, 159
(2014) 380–386.
8. GS Dhillon, Kaur S, Saurabh Jyoti Sarma, Brar SK. Integrated process for fungal citric acid
fermentation using apple processing wastes and sequential extraction of chitosan from
waste stream. Industrial Crops and Products, 50 (2013) 346–351.
xxxvi
9. G.S. Dhillon, S. Kaur, Saurabh Jyoti Sarma, S.K. Brar, V. Mausam, R.Y. Surampalli.
(2012). Recent development in applications of important biopolymer chitosan in
biomedicine, pharmaceuticals and personal care products. Current Tissue Engineering
(2013) 2 (1): 20-40.
10. G. Buelna, S. K. Brar, Y. L. Bihan, S. Gingras et Saurabh Jyoti Sarma. Le biohydrogène
par fermentation de matières résiduelles : une alternative énergétique prometteuse. Bio
trendance, 12-3 Janvier 2012.
11. Vinayak Pachapur, Saurabh Jyoti Sarma, Satinder Kaur Brar, Yann Le Bihan, Gerardo
Buelna, M. Verma. Use of co-culture in biohydrogen production – Insights and Challenges.
Biomass and Bioenergy (submitted).
12. Vinayak Pachapur, Saurabh Jyoti Sarma, Satinder Kaur Brar, Yann Le Bihan, Gerardo
Buelna, Mausam Verma. Energy balance for biodiesel derived crude glycerol from different
feedstock for biohydrogen production by dark fermentation. Journal of Renewable and
Sustainable Energy (submitted).
13. Krishnamoorthy Hegde, Niharika Chandra, Saurabh Jyoti Sarma, Current understandings
of risks and toxicity of engineered nanoparticles on microorganisms. Journal of Hazardous,
Toxic, and Radioactive Waste (submitted).
14. Krishnamoorthy Hegde, Niharika Chandra, Saurabh Jyoti Sarma, Satinder Kaur Brar,
Venkata Dasu Veeranki. Genetic engineering for sustainable biodiesel production. Applied
Microbiology and Biotechnology (submitted).
15. Krishnamoorthy Hegde, Saurabh Jyoti Sarma, Satinder Kaur Brar (2013). Dairy-derived
ingredients as nutraceuticals. Nutraceuticals and Functional Foods: Natural Remedy. Nova
Science Publishers, Inc. (published).
16. Krishnamoorthy Hegde, Saurabh Jyoti Sarma, Gurpreet Singh Dhillon, Satinder Kaur Brar
(2013). Nutraceuticals and liver health. Nutraceuticals and Functional Foods: Natural
Remedy. Nova Science Publishers, Inc. (published).
17. Krishnamoorthy Hegde, Rachna Goswami, Saurabh Jyoti Sarma, Venkata Dasu Veeranki
and Satinder Kaur Brar (2013). Environmental Hazards and Risks of Nanomaterials. In
Nanomaterials in the Environment, publisher: American Society of Civil Engineers (ASCE),
(in press).
18. Krishnamoorthy Hegde, Rachna Goswami, Saurabh Jyoti Sarma, Venkata Dasu Veeranki
and
Satinder Kaur Brar (2013).
Nano-ecotoxicology of
xxxvii
natural and
engineered
nanomaterials for other ecosystems. In Nanomaterials in the Environment, publisher:
American Society of Civil Engineers (ASCE), (in press).
19. Emna Chaabouni, Saurabh Jyoti Sarma, Fatma Gassara, Satinder K. Brar (2013). C3- C4
platform chemicals bioproduction using biomass. In Biotransformation of agro-industrial
wastes: high value biochemical; Springer, US (published).
20. Surinder Kaur, Gurpreet Singh Dhillon, Saurabh Jyoti Sarma, Satinder Kaur Brar, Kshipra
Misra, and Harinder Singh Oberoi. Waste Biomass: A Prospective Renewable Resource for
Development of Bio-Based Economy/Processes. In Biotransformation of agro-industrial
wastes: high value biochemical; Springer, US (published).
21. Krishnamoorthy Hegde, Niharika Chandra, Gurpreet Singh Dhillon, Saurabh Jyoti Sarma.
Fruit based functional beverages: properties and health benefits. Nova Science Publishers,
Inc. (published).
22. Satinder Kaur Brar, Saurabh Jyoti Sarma (2013). Bio-hydrogen production: a promising
strategy for organic waste management. Hydrology:Current Research. S5:e002. doi:
10.4172/2157-7587.S5-e002.
23. Satinder Kaur Brar, Saurabh Jyoti Sarma and Mausam Verma(2012). Energy recovery
from municipal and industrial wastes: How much Green? Hydrology:Current Research. Res
S5:e001.doi:10.4172/2157-7587.S5-e001.
24. Satinder Kaur Brar, Saurabh Jyoti Sarma, Emna Chaabouni (2012). Shelf-life of
biofertilizers: an accord between formulations and genetics. Journal of Biofertilizers and
Biopesticides. 3:e109.doi:10.4172/2155-6202.1000e109.
25. Ratul Kumar Das, Saurabh Jyoti Sarma, and Satinder Kaur Brar, Mausam Verma (2013)
Can production of biocontrol agent from waste biomass maximize waste utilization? Journal
of Biofertilizers & Biopesticides. 4:e113. doi: 10.4172/2155-6202.1000e113.
26. Ratul Kumar Das, Saurabh Jyoti Sarma, and Satinder Kaur Brar, Mausam Verma (2013)
Nanoformulation of Insecticides – Novel Products. Journal of Biofertilizers & Biopesticides.
doi: 10.4172/2155-6202.1000e120.
xxxviii
CONFÉRENCES
1. Saurabh Jyoti Sarma and Brar, S.K. Experimental feasibility of hydrogen production by
crude glycerol bioconversion. 1st Workshop Paraná-Quebec for Cooperative Research on
Bioenergy and Biotechnology, April 25th, 2012. UFPR, Curitiba, BRAZIL.
2. Saurabh Jyoti Sarma, Brar, S.K, Bihan, Y.L, Buelna, G. Biohydrogen production and valueaddition of spent medium: a sustainable method for biodiesel industry waste management.
International Conference on Green Chemistry and Sustainable Development. 29th to 31st
July 2014, Barcelona, SPAIN.
3. Saurabh Jyoti Sarma, Satinder Brar, Yann LeBihan, Gerardo Buelna. Integrated
biohydrogen and organic acid production- a green technology for biodiesel industry waste
management. The 2014 ASABE and CSBE | SCGAB Annual International Meeting. July 1316, 2014, Montreal, CANADA.
4. Saurabh Jyoti Sarma, Satinder Brar, Yann LeBihan, Gerardo Buelna. Integrated
biohydrogen and phosphate solubilizer production using biodiesel industry waste. 28e
CONGRÈS ANNUEL DE L’AQSSS. May 26 -29, 2014, Victoriaville, CANADA.
5. Vinayak Pachapur, Saurabh Jyoti Sarma, Ratul Das, Satinder Brar, Yann LeBihan,
Gerardo Buelna, Mausam Verma. Energy balance and greenhouse gas emissions
evaluation on biohydrogen production from crude glycerol by dark fermentation. The 2014
ASABE and CSBE | SCGAB Annual International Meeting. July 13-16, 2014, Montreal,
CANADA.
xxxix
CHAPITRE 1
SYNTHÈSE
Chapitre 1. Synthèse
PARTIE I. PRODUCTION D'HYDROGENE PAR GLYCÉROL BRUT
BIOCONVERSION-REVUE DE LA LITTÉRATURE
1.1. Introduction
Les combustibles fossiles sont les principales sources d'énergie naturelles, qui couvrent
près de 80% de la demande mondiale d'énergie (Ni et al., 2006). Cependant, le stock
mondial de carburant fossile s’épuise rapidement et la combustion de combustibles
accroît les impacts négatifs mondialement sur le climat (Aksoy, 2011, Gnana Kumar et
al., 2010). Une telle situation a déclenché une recherche rapide des sources d'énergie
de remplacement dans le monde entier. “L'énergie durable pour tous” est une initiative
des Nations Unies et “doubler la part des énergies renouvelables dans le mix
énergétique mondial” est l'un de ses trois objectifs principaux à remplir d'ici 2030
(http://www.unfoundation.org/what
-we-do/issues/energy-and-climate/clean-energy-
development.html. consulté le 8 mai 2013). Cette initiative est l'un des exemples de
l'intérêt de la communauté mondiale vers le développement de sources d'énergie
renouvelables pour la sécurité énergétique de l'avenir. Les biocarburants étant une
source d'énergie renouvelable ils peuvent être un substitut approprié aux combustibles
fossiles.
Le biodiesel est un des biocarburants les plus couramment étudiés et il peut être produit
par trans-estérification d'acides gras naturels (Muniyappa et al., 1996). En raison de la
disponibilité des matières premières, la mise en œuvre simple des technologies pour sa
production et son rôle dans le bilan des gaz à effet de serre (GES); la production
mondiale de biodiesel est en augmentation rapide. À l'heure actuelle (2013), la
production mondiale de biodiesel est d'environ 30 milliards de litres, et il devrait
atteindre
42
milliards
de
litres
d'ici
2021
(http://www.fao.org/fileadmin/templates/est/COMM_MARKETS_MONITORING/Oilcrops
/Documents/OECD_Reports/biofuels_chapter.pdf. Consulté le 9 mai 2013.). En outre, la
production de biodiesel a été promue par les différents gouvernements par diverses
subventions et politiques. Le programme de norme sur les carburants renouvelables
révisé (RFS2) a été mis au point, selon lequel, à partir de 2012, chaque année un
minimum de 1 milliard de gallons de biodiesel sera mélangé dans le carburant diesel et
3
il faudra environ 36 milliards de litres de carburant renouvelable d'ici 2022 (http: / /
www.biodieselmagazine.com/articles/4080/report-12-billion-gallons-of-biodiesel-by2020. consulté le 27 décembre 2011). De même, le Canada est un important
producteur de biodiesel en Amérique du Nord et le gouvernement du Canada a fixé un
objectif de réduction des GES de 17 pour cent d'ici 2020 et de 2% de contenu
renouvelable
sera
obligatoire
pour
le
diesel
(http://www.ec.gc.ca/default.asp?lang=En&n=714D9AAE-1&news=BDE26528-CBCB4008-949D-CDFDFBD054FB. consulté le 27 décembre 2011) (Règlement modifiant le
Règlement sur les carburants renouvelables, de l'environnement canadien loi sur la
protection,
1999,
ministère
de
l'Environnement,
Canada.
https://tsapps.nist.gov/notifyus/docs/wto_country/CAN/corrigenda/pdf/CAN311_add_3
(anglais). pdf. Consulté le 27 décembre 2011).
Au cours du processus de production du biodiesel, un grand volume de glycérol brut
(GB) est produit comme sous-produit. En poids, la proportion de glycérol est de 10% de
biodiesel produit (Selembo et al., 2009a, Yazdani et al., 2007). Le glycérol est une
matière première importante pour l’alimentation, l’industrie pharmaceutique et
cosmétique, et autres processus de fabrication (Guerrero - Pérez et al., 2009).
Toutefois, en raison de la présence d'une grande quantité d'impuretés (méthanol,
savon, sels, huiles et matières organiques solides) (Chi et al., 2007, Ngo et al., 2011,
Selembo et al., 2009b); le glycérol brut (GB) ne peut pas être directement utilisé dans
les industries alimentaires ou pharmaceutiques et sa purification est très coûteuse
(Escapa et al., 2009). En outre, le GB, considéré comme un déchet, s’il est directement
rejeté dans l'environnement sans traitement approprié, pourrait avoir des conséquences
néfastes (da Silva et al., 2009, Selembo et al., 2009a). Comme alternative, le GB
contenant des déchets d'effluent d’usine de production de biodiesel peut être valorisé
comme un substrat moins coûteux pour la production de différents produits à valeur
ajoutée (PHVA), tels que 1, 3 -propanediol, l'éthanol, et les aliments pour animaux, (da
Silva et al., 2009, Wang et al., 2001). Toutefois, en raison de la présence d'impuretés,
la récupération et purification des PHVA produits sont également complexes et
coûteuses (Pachauri et al., 2006). Par contre, le GB peut être utilisé comme substrat
pour la production d'hydrogène, dans ce cas, le produit recherché (H2) est
4
automatiquement séparé du milieu et il peut être récupéré en utilisant des techniques
simples.
La production de H2 en utilisant le GB généré par l’industrie du biodiesel a déjà été
étudiée par plusieurs chercheurs (Ito et al., 2005, Ngo et al., 2011, Selembo et al.,
2009a). Il est établi que l’H2 a un contenu très élevé en énergie et il peut être utilisé
comme biocarburant (Ngo et al., 2011). La chaleur de combustion de l'hydrogène est 285,8 kJ/mol et il a une teneur en énergie de 142,9 kJ/g (disponible à partir de:
http://www.creative-chemistry.org.uk/gcse/documents/Module21/N-m21- 10.pdf).
En outre, si l’H2 est utilisé comme combustible, le seul sous-produit généré est l'eau
(Selembo et al., 2009a). Par conséquent, compte tenu de la préoccupation croissante
sur le réchauffement climatique, l’H2 peut être considéré comme le carburant le plus
propre pour l'avenir. Par conséquent, la bioconversion du GB en hydrogène est une
approche avec un avenir prometteur. L’H2 peut aussi être réalisé par différentes
techniques chimiques, tels que le reformage du gaz naturel (Chen et al., 2008, Roh et
al., 2010.); l’oxydation partielle de l'huile lourde (Liu, 1999) et la gazéification du
charbon (Stiegel et al., 2006). Contrairement à la production de l’H2 biologique, toutes
ces techniques utilisent des combustibles fossiles comme matière première pour la
production d'hydrogène et ils ne sont pas considérés comme des processus durables.
Ainsi, dans le cadre de l'intérêt mondial pour le développement des sources d'énergie
renouvelables, la production de biohydrogène par la bioconversion du GB peut être
considéré comme une technologie prometteuse.
La fermentation sombre, la photofermentation, ou les deux étapes en série et
l'application de l'électrolyse microbienne sont les différentes méthodes couramment
utilisées pour la production biologique d'hydrogène (Call et al., 2008, Meher Kotay et al.,
2008). Jusqu'à ce jour, diverses stratégies ont été appliquées pour la production de H2
par bioconversion du GB dont la fermentation sombre; celle-ci est le processus le mieux
étudié. Ngo et al (2011) ont rapporté une production de 2,73 ± 0,14 mol-H2/mol glycérol
par Thermotoga neapolitana (Ngo et al., 2011). De même, Fernandes et al (2010) ont
rapporté une production de 200 mL -H2/g COD (Fernandes et al., 2010). Dans une autre
étude similaire, Marques et al (2009) ont utilisé Enrerobacter aerogenes et les auteurs
5
ont rapporté une production de 2,5 dm3- H2/dm3 milieu de culture (Marques et al.,
2009). Cependant, dans tous ces cas, malgré un rendement très élevé en hydrogène,
l’application technologique a encore beaucoup de problèmes à surmonter pour une
application industrielle potentielle.
Dans cette recherche, une section de revue de la littérature présente une étude
systématique et comparative des rapports actuellement disponibles sur la production de
bio-hydrogène à partir de glycérol brut. Cette section cible également les orientations
futures de recherche.
Tout d'abord, les caractéristiques du GB généré à partir du procédé de fabrication du
biodiesel sont examinées suivies du potentiel de production du biohydrogène. Ensuite,
divers procédés de pré-traitement, les types de bioréacteurs utilisés, ainsi que le
potentiel de bioconversion du glycérol par différents micro-organismes ont été abordés.
Finalement, diverses stratégies pour améliorer la production d'hydrogène ont été
proposées. Pour une présentation plus détaillée, l'article scientifique présenté dans le
chapitre 2 de cette thèse (Sarma et al., 2012) peut être consulté.
1.2. Glycérol: sous-produit de la production du biodiesel
1.2.1. Transestérification d'huiles végétales et des graisses animales: Le glycérol
est un sous-produit du processus de production du biodiesel. Pour la production du
biodiesel, de façon classique, les triglycérides, telles que les huiles végétales et les
graisses animales sont mélangées avec du méthanol dans un réacteur. De l'hydroxyde
de sodium est ajouté comme catalyseur de la réaction et le mélange est agité et chauffé
à la température d'ébullition du methanol (Kim et al., 2004, Muniyappa et al., 1996,
Schuchardt et al., 1998). Lors de l’arrêt de l'agitation, le mélange réactionnel se sépare
en deux phases. La couche supérieure est l'ester méthylique d'acides gras soit le
biodiesel et la couche inférieure est le GB contenant du méthanol n'ayant pas réagi, le
sel et les substances solides présentes dans les matières premières. La Figure 2.1.1
représente une vue schématique de transestérification et le processus de production du
biodiesel. La Figure 2.1.2 montre l'équation chimique pour la production de glycérol par
transestérification de triglycérides (Muniyappa et al., 1996). La phase de GB contient
généralement près de 75 % de glycérol (Muniyappa et al., 1996). Toutefois, le contenu
6
de glycérol dans les déchets de production du biodiesel peut varier d'une usine à l’autre
(Tableau 2.1.1).
1.2.2. Impuretés présentes dans le glycérol brut: En raison de sa haute teneur en
énergie, différents chercheurs du monde entier ont étudié la bioconversion du GB en
produit à valeur ajoutée (Ito et al., 2005, Mu et al., 2006, Ngo et al., 2011, Sakai et al.,
2007, Selembo et al., 2009a, Selembo et al., 2009b). Ils ont caractérisé le GB
provenant des usines de fabrication du biodiesel pour obtenir une vue d’ensemble des
impuretés présentes. Chi et al (2007) ont rapporté que les impuretés principalement
présentes dans GB sont le savon, les acides gras libres, le méthanol, les triglycérides
qui n'ont pas réagi, les diglycérides et monoglycérides (Chi et al., 2007) (Tableau 2.1.1).
Le GB du processus de fabrication du biodiesel peut également contenir une petite
quantité de l'huile utilisée pour la transestérification (Chi et al., 2007) et d'autres
impuretés présentes dans cette huile.
1.2.3. Problèmes environnementaux reliés aux rejets du glycérol provenant de la
production de biodiesel: Comme mentionné ci-dessus, le GB généré à partir des
usines de fabrication du biodiesel peut contenir jusqu'à 25% (p/p) de méthanol (Ito et
al., 2005). À partir de différentes études, il a été conclu que les déchets du biodiesel
contenant du méthanol sont inhibiteurs de la croissance microbienne (Athalye et al.,
2009, Chi et al., 2007) et peuvent être toxiques pour l'environnement, s'ils sont libérés
sans traitements appropriés. L'excès de méthanol présent dans le GB peut contaminer
l'eau souterraine. De même, en raison de l'addition de NaOH dans le processus de
fabrication du biodiesel, le pH des déchets produits est généralement trop élevé et doit
être neutralisé avant l'élimination des déchets. De plus, les eaux usées à salinité et
alcalinité élevées peuvent prendre plus de temps pour être dégradées par les
microorganismes indigènes du milieu récepteur.
1.3. Glycérol: un substrat pour la production de bio-hydrogène
1.3.1. Glycérol comme matière première pour la production d'hydrogène: Le
glycérol est un substrat qui peut être utilisé par de nombreux microorganismes pour leur
croissance et qui a été largement étudié pour son potentiel de production d'hydrogène.
Le contenu énergétique du glycérol pur est de 19,0 MJ/kg; cependant, il est de 25,30
7
MJ/kg pour le GB en raison de la présence de méthanol et des traces de biodiesel
(Disponible
à
partir
de:
http://www.esru.strath.ac.uk/EandE/Web_sites/06-
07/Biodiesel/experiment.htm). Cette haute teneur en énergie du glycérol brut indique un
fort potentiel comme substrat pour la production d'hydrogène. En outre, contrairement à
la plupart des matériaux des déchets cellulosiques, il ne nécessite pas de prétraitement
supplémentaire pour le rendre disponible pour les micro-organismes produisant de
l'hydrogène. Également, des substrats tels que le lactosérum et la mélasse sont en forte
demande en raison de leur large éventail d'applications dans les fermentations
industrielles. En contrepartie, des substrats tels que les déchets alimentaires
contiennent généralement des matières solides d'origines différentes et ont besoin de
broyage et de mélange approprié avant de les soumettre à la fermentation. Ainsi, pour
la production d'hydrogène à grande échelle, le GB semble être le support idéal sans
contraintes de pré-traitements majeurs. Différents rapports sur la production
d'hydrogène par bioconversion du glycérol pur et brut ont été résumées dans le Tableau
2.1.3.
1.3.2. Métabolisme du glycérol et de la production d'hydrogène: Les métabolismes
oxydatifs et réducteurs du glycérol sont connus pour certaines espèces telles que
Klebsiella, Citrobacter, Clostridium, et Enterobacter (Drożdżyńska et al., 2011). Comme
le montre la Figure 2.1.4, dans la voie oxydative, le glycérol est d'abord converti en
dihydroxyacétone sous l'activité catalytique de la glycérol déshydrogénase. Dans la
prochaine étape, la dihydroxyacétone est phosphorylé par l'enzyme glycolytique, la
dihydroxyacétone kinase. Enfin, le produit phosphorylé est métabolisé par la glycolyse
(Daniel et al., 1995, Drożdżyńska et al., 2011, Luers et al., 1997). Dans la voie de la
réduction, le glycerol est finalement converti en PD (1,3 -propanediol) par l'intermédiaire
de la production du produit 3-hydroxypropionaldéhyde.
D'après la Figure 2.1.4, on peut voir que pendant le métabolisme oxydatif du glycérol, le
pyruvate est formé comme produit intermédiaire; qui, à son tour, peut être métabolisé
en divers produits finaux tels que l'éthanol, le butanol, l'acétone, l'acétate, le butyrate et
le lactate (da Silva et al., 2009, Drożdżyńska et al., 2011). Dans la plupart des voies de
bioconversion du glycérol, ainsi que les différents métabolites, de l’H2 est également
8
produit au cours du métabolisme oxydatif. À partir des équations stœchiométriques
présentées dans la Figure 2.1.5, on peut conclure que la production du produit final très
réduit s'accompagne d'un rendement de de production faible de H2. Cette réaction peut
être vue à partir de la Figure 2.1.6 où le pyruvate est décomposé en acétyl -CoA par
l'intermédiaire d'une réduction de la ferrédoxine (Fd) catalysée par la pyruvate
ferrédoxine oxydoréductase. La ferrédoxine réduite (Fd) est ensuite oxydée par une
hydrogénase qui reproduit la Fd oxydée et de l'hydrogène gazeux (Mathews et al. 2009,
Nath et al., 2004). Une quantité supplémentaire d'hydrogène peut être produite par la
glycolyse lorsque le NADH est oxydé par la réduction de la Fd et NADH-ferrédoxine
réductase (Mathews et al., 2009, Vardar-Schara et al., 2008). Théoriquement, un
maximum de 4 moles de H2 peuvent être produites par mole de glucose lorsque
l'acétate est le produit final de fermentation. Cependant, seulement deux moles de H2
par mole-glucose peuvent être générées lors de la production du butyrate et autres
produits finaux réduits tels que les alcools et l'acide lactique (Levin et al ., 2004,
Mathews et al., 2009).
1.4. Prétraitement du glycérol brut
1.4.1. Importance du prétraitement du glycérol brut: Tel que mentionné
précédemment, le processus de transestérification pour la production de biodiesel
implique un mélange de graisses animales et/ou végétales avec du méthanol et de
l'hydroxyde de sodium comme catalyseur (Kim et al., 2004, Muniyappa et al., 1996,
Schuchardt et al., 1998). Par conséquent, en plus du glycérol, un rejet typique de
production de biodiesel peut contenir des concentrations élevées en sels, en méthanol,
en savon et en précipités solides (Athalye et al., 2009, Yazdani et al., 2007), ce qui peut
affecter la croissance microbienne lors de la bioconversion (Chi et al., 2007, Ngo et al.,
2011). Afin d'éviter de tels incidents, le GB doit être prétraité avant d'être utilisé comme
matière première pour la production d'hydrogène.
1.4.2. Différentes méthodes de prétraitements du GB: Pour utiliser le GB comme
matière première pour la bioconversion, le méthanol peut être retiré à l'autoclave
(Athalye et al., 2009, Ethier et al., 2011). Selon Pyle et al (2008), le méthanol s'évapore
à 65 °C, d'où le processus de passage à l'autoclave durant 15 min à 121°C afin
9
d'éliminer une quantité importante de méthanol à partir du GB (Pyle et al., 2008). Dans
une récente recherche sur la production d'hydrogène à partir de glycérol brut, Ngo et al
(2010) ont rapporté l'élimination du méthanol et de l'éthanol à l'évaporateur rotatif à 45
°C, où les solides ont été récoltés par centrifugation à 15 000 rpm pendant 15 min (Ngo
et al., 2011). Le savon est l'autre impureté majeure présente dans le GB, qui peut être
éliminé par précipitation à partir du milieu liquide à l’aide d’un ajustement du pH
(Athalye et al., 2009, Ethier et al., 2011). Ainsi, diverses méthodes d'élimination des
impuretés ont été évaluées et la possibilité d'utiliser une seule méthode d'élimination
collective pour toutes les principales impuretés présentes dans le GB devraient être
explorées.
1.5. Génie biologique pour la production d'hydrogène
1.5.1. Évaluation de différents types de réacteurs pour la production de biohydrogène: Pour évaluer la production d'hydrogène en utilisant différents déchets
organiques, y compris le GB généré dans les usines de production de biodiesel,
différents types de réacteurs ont été utilisés. Le bioréacteur utilisé à cette fin peut être
une petite fiole à sérum de 10 mL de volume de travail ou un fermenteur de 2,5-L. Un
résumé des types de bioréacteurs utilisés pour la production d'hydrogène a été
présenté dans le tableau 2.1.4. Dans un rapport sur la production d'hydrogène à partir
de déchets de la transformation du biodiesel, Ito et al (2004) ont indiqué que les
déchets de production de biodiesel contenant du glycerol doivent être dilués avec un
milieu synthétique pour augmenter le taux d'utilisation du glycérol. Ils ont rapporté que
la production de H2 diminue avec une augmentation de la concentration des déchets du
biodiesel (Ito et al., 2005). Par conséquent, pour la bioconversion d’un grand volume de
GB dilué, un système continu peut être plus approprié qu'un système de traitement en
mode discontinu.
D’ailleurs, au niveau de la recherche d’informations il y a moins
d’études qui traite des systèmes en lots.
1.5.2. Paramètres importants du procédé et leur niveau optimal:
Selon le tableau 2.1.4, il est clair que la production d'hydrogène microbienne a été
étudiée en utilisant différents types de réacteurs et les paramètres optimaux des
procédés ont été choisis pour des cas spécifiques. La gamme de température utilisée
10
par différents chercheurs pour la production d'hydrogène varie de 25º C à 75º C
(Escapa et al., 2009, Ngo et al., 2011). Pour le pH des valeurs de 4,5 à 7,5 ont été
signalées (Fang et al., 2006, Ngo et al., 2011). Les paramètres du procédé peuvent
varier en fonction du mode de fonctionnement ou les types de microorganismes utilisés
dans le procédé. Par conséquent, une température ou un pH particulier ne peuvent pas
être considérées comme optimales pour le procédé de production d'hydrogène dans
son ensemble.
1.6. Microorganismes utilisés pour la bioconversion du glycerol
1.6.1. Microorganismes utilisés actuellement pour la bioconversion du glycérol.
Comme le glycérol a été utilisé pour la production de différents produits; divers
microorganismes ont été évalués pour leur capacité de bioconversion du glycérol.
Différents microorganismes utilisés actuellement pour la bioconversion du GB ont été
répertoriés dans le Tableau 2.1.5. À haute température, la production d'hydrogène est
plus exergonique et les microorganismes hyper thermophiles présentent une résistance
à des pressions partielles d'hydrogène élevées (van Niel et al., 2003). L'un des
avantages de la fermentation à des températures extrêmes, c'est que le procédé est
moins sensible aux contaminations, mais d'autre part, il est plus complexe de réaliser
une relation économique positive entre l'énergie utilisée pour chauffer et maintenir le
réacteur à des températures élevées et la production de H2. En outre, les bactéries
anaérobies thermophiles extrêmes se développent habituellement en faibles densités
résultant d’un taux de production faibles. Les chercheurs ont étudié la bioconversion du
glycérol en utilisant la monoculture ou un consortium microbien mixte. Phowan et al
(2010) ont démontré que la co-culture de C. butyricum TISTR 1032 et E. aerogenes
TISTR 1468 était capable de réduire la phase de latence de la production d'hydrogène
(Phowan et al., 2010). Comme il est montré dans le Tableau 2.1.5, Enterobacter
aerogenes est l'organisme le plus étudié pour la production d'hydrogène à l'aide du GB.
Les cultures microbiennes mixtes à partir de sources environnementales sont les autres
inoculums les plus utilisés pour la bioconversion du GB. Cependant, certaines
contraintes au niveau de la stabilité du processus existent et doivent être considérées
11
surtout si des eaux usées industrielles avec des variations de composition sont
utilisées.
1.6.2. Génie génétique et métabolique pour l'amélioration de la production
d'hydrogène: Le génie génétique et métabolique peut être considéré comme un outil
potentiel d'amélioration de la production d'hydrogène par bioconversion du GB. Maeda
et al (2007) ont rapporté qu'une souche de E. coli recombinant est capable d’exprimer
une enzyme bidirectionnel la [NiFe]-hydrogénase de Synechocystis sp. PCC 680
(Maeda et al., 2007). Une augmentation de la production d'hydrogène de facteur de 41
fois par rapport au type sauvage a été signalé pour la souche recombinante. Chittibabu
et al (2006) ont également mis au point une souche génétiquement modifiée de E. coli
contenant le gène [FeFe]-hydrogénase de Enterobacter cloacae IIT BT- 08. Avec cette
souche génétiquement modifiée, un rendement de 3.12 mol H2 par mole de glucose a
été obtenu, ce qui était beaucoup plus élevé que le type sauvage (Chittibabu et al.,
2006). Ainsi, un certain nombre de succès ont été obtenus à partir des tentatives de
génie génétique pour améliorer la production de biohydrogène. Toutefois, le potentiel
de production d'hydrogène d'une souche recombinante, en utilisant différents déchets
organiques en tant que substrat n'est pas encore documenté. Dans une étude récente,
Mathews et Wang (2009) ont suggéré que, pour accroître la viabilité économique de la
production d'hydrogène, différentes voies d'utilisation du substrat doivent être conçues
dans des organismes produisant de l'hydrogène (Mathews et al., 2009). Par
conséquent, une souche microbienne issue du génie génétique et qui présente une
bioconversion efficace du GB peut améliorer la viabilité économique de la production de
biohydrogène. Récemment, en utilisant l'évolution adaptative et la mutagenèse
chimique, suivie par l'expérience de croissance sur le glycérol, Hu et Wood (2009) ont
mis au point une amélioration de la souche d'Escherichia coli (HW2), qui produit 20 fois
plus d'hydrogène dans du glycérol contenant un supplément (0,68 ± 0,16 mmol-H2 L-1 h
-1
) (Hongbo Hu et al., 2010). Shams Yazdani et Gonzalez (2008) ont également décrit
une souche recombinante d 'Escherichia coli capable de bioconvertir du glycérol en
éthanol et en hydrogène (Yazdani et al., 2007).
12
PARTIE II. PROBLÈMES, HYPOTHÈSE, OBJECTIFS ET ORIGINALITÉ
2.1. Problèmes
À partir de la revue de la littérature (Sarma et al., 2012) et l'analyse de la faisabilité
technico-économique de la production de biohydrogène (Sarma et al., 2013) présentée
au chapitre 2, les problématiques suivantes ont été soulevées
2.1.1. Coût des composantes et média supplémentaires
Le coût de fonctionnement des processus récemment évalués pour la production
d'hydrogène par bioconversion du GB est très élevé et, par conséquent, ils ne sont pas
économiquement viables pour des applications commerciales. Dans l'analyse de
faisabilité, il a été mis en évidence que le coût des composantes des milieux est
d'environ 82% du coût total du procédé. Ceci est dû au fait qu'une grande quantité de
milieu nutritif (50-400 L/kg GB) doit être ajoutée pour diluer le GB à une très faible
concentration de départ pour sa bioconversion. D'autre part, le coût de développement
de l'inoculum est de près de 27% du coût total de production.
2.1.2. Effet inhibiteur des impuretés dans le GB (comme le savon, le méthanol,
NaCl) sur la production de H2
Le GB n'est pas un substrat pur pour la production d'hydrogène et il peut contenir une
variété d'impuretés. Du méthanol (23,4 à 37,5% p/p) (Tang et al., 2009, Thompson et
al., 2006), le sel (3,12% p/p) (http://rendermagazine.com/articles/2008-issues/2008 august/2008-08-crude-glycerin / (consulté le 03_09_2012) et du savon (de 20,5 à
31,4% p/p) (Shengjun Hu et al., 2012) sont trois des principales impuretés présentes
dans le GB et ils pourraient avoir certains effets sur la production d'hydrogène
(Venkataramanan et al., 2012, Yang et al., 2012). Les impuretés, telles que le méthanol
et le savon, peuvent également être utilisées comme sources de carbone, leur
présence ou leur absence dans les milieux pourraient affecter les performances du
processus. Par conséquent, l'effet de ces trois impuretés du GB sur la production
d'hydrogène doit être évalué et des stratégies appropriées devraient être élaborées
pour atténuer leurs effets inhibiteurs.
13
2.1.3. Co-production d'un grand volume de déchets liquides pendant la
production de biohydrogène
Sur la base de notre estimation (Sarma et al., 2013), près de 50-400 L de déchets sont
générés lors de la production de H2 à partir d'un kg de GB. Compte tenu du volume
important de milieu, il est à noter que la gestion durable de ces déchets liquides sera
nécessaire.
2.1.4. Effet inhibiteur du pH sur la production du H2 en fermentation sombre
Lors de la production de biohydrogène par fermentation sombre, une gamme d'acides
organiques sont produits et ils sont généralement considérés comme des déchets
(Shida et al., 2009). En outre, la production d'acide organique peut consommer une
partie importante du substrat disponible pour la production de H2 et diminuer le
rendement et aussi abaisser le pH du milieu ce qui peut inhiber le processus de
fermentation. Par conséquent, il est considéré comme le principal frein technicoéconomique
de
la
fermentation
sombre
pour
produire
du
H2
(http://www.slideshare.net/kumarhukkeri/finalppt-em-1 (consulté le 02/03/2013)).
2.1.5. Auto inhibition de la production par accumulation de H2
Il est bien connu que le mode de traitement peut fortement influencer le rendement en
hydrogène. Dans un procédé discontinu, l’H2 s'accumule dans l'espace de tête du
réacteur pour augmenter la pression partielle de H2, qui, à son tour, peut inhiber la
production de H2 (Oh et al., 2009, Van Andel et al., 1985). Avec l'hydrogène, le CO2 est
produit et même l'accumulation de CO2 a un effet négatif sur la production d'hydrogène
(Oh et al., 2009, Park et al., 2005). Avec l'augmentation de la concentration en
hydrogène, la voie métabolique est décalée vers la production de solvants et augmente
la production de produits réduits, tels que le butanol, le lactate ou de l'acétone, résultant
en une diminution de la production d'hydrogène (Junghare et al., 2012, Levin et al.,
2004). Dans une étude utilisant Clostridium butyricum TM-9A, Junghare et al (2012) ont
montré qu’il est possible d’augmenter la production d'hydrogène de 2,6 fois par la
diminution de la pression partielle de 33,9 à 10,13 kPa (Junghare et al., 2012).
14
2.2. Hypothèse
Le projet de recherche comprend différentes hypothèses:
2.2.1. Le GB est un sous-produit de la production de biodiesel par transestérification. Sur
la base de la nature de la charge, le catalyseur et les conditions de réaction utilisées
pour ce procédé, la composition du GB pourrait varier. Le processus de production de
biodiesel génère le GB qui nécessite une bonne caractérisation avant son utilisation
pour la production d'hydrogène.
2.2.2. Le GB est une bonne source de carbone et il a le potentiel pour soutenir la
croissance microbienne. Par conséquent, E. aerogenes, la bactérie choisie pour la
présente étude doit se développer sur le GB en l'utilisant comme seul substrat et
produire le H2. Si elle peut être cultivée avec succès, aucun nutriments supplémentaires
ne sera nécessaire et le coût des processus de production de H2 pourrait être réduit. En
outre, si la biomasse produite pendant la fermentation pourrait être ré-utilisée dans le
processus ultérieur,
soit
comme
inoculum ou
comme
source
de
nutriment
supplémentaire, une nouvelle réduction du coût de traitement pourrait être possible.
2.2.3. Comme mentionné dans la section de revue de la littérature, le GB est
généralement complété par des suppléments de milieu synthétique pour une meilleure
production de H2. Dans ce contexte, les déchets riches en éléments nutritifs, tels que
les boues secondaires provenant des usines de traitement des eaux usées, les eaux
usées d’abattoir et les rejets de brasserie entre autres pourraient être évalués en tant
que remplacement des composantes de milieux synthétiques.
2.2.4. Outre le glycérol, le GB contient une série d'autres composés, tels que le savon, le
méthanol, NaCl etc. Selon la revue de la littérature, il est évident que certains de ces
composés pourraient avoir un effet négatif sur la croissance microbienne alors que
certains d'entre eux pourraient avoir un apport bénéfique pour le processus. Ainsi, une
étude doit être réalisée en utilisant le glycérol pur en tant que substrat et les différents
composantes du GB pourraient être ajoutées de façon externe afin d'évaluer leur effet
sur la production de H2. Pour réduire le nombre d'expériences nécessaires pour cette
15
recherche et avoir une idée claire des effets interactifs des différentes composantes,
l'approche de la méthodologie de réponse de surface pourrait être utilisée.
2.2.5. Le savon est l'une des principales impuretés présentes dans le GB et il est
reconnu pour avoir des effets négatifs sur la croissance microbienne. En augmentant la
salinité du GB, le savon peut être précipité et être éliminé par centrifugation. Ainsi, le
NaCl peut être utilisé pour l'enlèvement du savon et son effet sur la production
d'hydrogène pourrait être étudié. De même, le Mg2+ peut réagir avec des molécules de
savon et les inactiver pour former de l'écume. Ainsi, l'addition de MgSO4 pour un milieu
à base de GB pourrait détruire les propriétés fonctionnelles du savon et réduire son
effet inhibiteur et améliore la production d'hydrogène.
2.2.6. Le fer est un cofacteur de l'hydrogénase, l'enzyme clé responsable de la
production d'hydrogène. Par conséquent, la production d'hydrogène peut être améliorée
par l’addition d'un composé contenant du fer dans le milieu utilisé à cet effet. Les
composés, tels que le FeCl2 et FeSO4 ont été rapportés comme ayant un potentiel
d’améliorer la production d'hydrogène. Dans ce contexte, le citrate ferrique peut être
évalué en tant que supplément pour améliorer la production d'hydrogène à partir du GB.
En plus d'être une source de fer, il est capable d'agir comme une source de carbone
supplémentaire et facilement accessible pour le métabolisme et par conséquent, on
peut prévoir qu'il y aura de meilleurs rendements que le FeCl2 et FeSO4,
traditionnellement utilisé dans ce but.
2.2.7. Pendant la production d'hydrogène en fin de fermentation, essentiellement des
acides organiques accumulent dans le milieu. Ces acides peuvent diminuer le pH du
milieu, initier des changements métaboliques et réduire la perfomance du processus.
Par conséquent, un système de bioréacteur de séparation à deux phases peut être
développé pour éliminer in situ les acides organiques générés lors de la fermentation.
Dans ce cas, les acides organiques produits au cours de la fermentation seront
solubilisés dans la phase organique, de sorte que les bactéries présentes dans la
phase aqueuse ne subiront pas l'effet néfaste de ces acides.
2.2.8. Le mode de fermentation adopté pour la production d'hydrogène a une forte
influence sur les rendements de production. Dans le cas du procédé classique
16
discontinu fermé, le gaz produit est accumulé dans l’espace gazeux du réacteur avec
une augmentation graduelle de la pression partielle du H2. Cependant, l'augmentation
de la pression partielle du H2 est inhibitrice pour le procédé. La production de H2
pourrait être améliorée par une libération continue du gaz produit. Également, une
fermentation continue presente certains avantages par rapport à une fermentation par
lots. En particulier, lorsque l'accumulation de sous-produits est inhibitrice pour le
procédé, ceux-ci sont évacués en continu. Par conséquent, un bioréacteur de 7,5 L a
été utilisé pour la production continue d'hydrogène en libérant de façon continue le gaz
produit. Cependant, dans ce cas, la surveillance de la concentration du gaz produit sera
difficile. Par conséquent, un capteur d'hydrogène peut être utilisé pour la surveillance en
temps réel de la production de H2.
2.2.9. Conformément à l'estimation, lors de la production d'hydrogène par fermentation
pour 1 kg de GB, un volume aussi élevé que 50 à 400 litres d'effluents liquides
contenant une gamme d'acides organiques seront générés. Fait intéressant, ils existent
sur le marché des bio-engrais commerciaux (PSB) contenant des acides organiques qui
peuvent chélater les cations de phosphates insolubles (comme Ca3(PO4)2) présents
dans le sol pour solubiliser le phosphate et le rendre disponible pour les plantes. Ainsi,
la capacité de solubilisation du phosphate du rejet liquide de fermentation du GB
pourrait être évaluée. En cas de succès, une plante modèle pourrait être utilisée pour
vérifier son efficacité comme une alternative potentielle de PSB.
2.3. Objectifs
L'objectif principal de cette étude est de renforcer la production d'hydrogène par
bioconversion du glycérol brut en réduisant les coûts de fermentation et utiliser de façon
durable les déchets générés au cours du processus afin d'offrir une solution " zéro
déchet " pour la gestion du glycérol brut.
Sur la base de ce qui précède, une revue de la littérature et des recherches
préliminaires ont été réalisées et ont permis d’élaborer les objectifs spécifiques
suivants :
17
2.3.1. Caractérisation du glycérol brut
Le GB est caractérisé pour la teneur en glycérol, le savon total, la quantité totale de
carbone, l'azote total et les différents éléments, notamment le Fe, Mg, S, P, Ni, Mn, Cu,
Zn, etc.
2.3.2. Production de biohydrogène à partir de la bioconversion du GB provenant
d’une usine de production de biodiesel : évaluation technico-économique
Sur la base des données disponibles dans la littérature sur la bioconversion du GB et la
production biologique d'hydrogène, une étude de faisabilité technico-économique du
processus sera effectuée.
2.3.3. Évaluation de GB en tant que seule substrat pour la production
d'hydrogène et valorisation des déchets dans le processus ultérieur
Afin de déterminer le potentiel réel du GB comme matière première pour la production
de biohydrogène, et de déterminer l'effet de la concentration initiale de GB sur la
production de H2, différentes concentrations de GB seront testées comme substrat. De
plus, les déchets (comme la biomasse) générés au cours du processus seront réutilisés
(soit comme inoculum ou comme source d' éléments nutritifs) dans le processus
ultérieur.
2.3.4. Screening de différents déchets en tant que source de nutriments
supplémentaires
Similaire à l'objectif 2, si les déchets tels que les boues des stations d'épuration des
eaux usées, les eaux usées d’abattoir ou les déchets de brasserie pouvaient être
utilisés comme remplacement des suppléments synthétiques coûteux, une réduction
des coûts du procédé de fermentation serait possible. Par conséquent, une gamme de
ces matériaux sera testée pour leur application possible dans le processus de
bioconversion du GB.
18
2.3.5. Étude de l'effet des différentes impuretés du glycérol brut sur la production
d'hydrogène et de l'utilisation du glycérol par E. aerogenes utilisant du glycérol
pur comme substrat
En utilisant la méthodologie de surface deréponse , l’effet individuel et interactif de trois
principales impuretés de GB (savon, méthanol, NaCl) sera évalué. À cet effet, des
expériences de production de H2, en utilisant le glycérol pur en mélange avec des
quantités définies (selon la conception expérimentale) des impuretés seront utilisées
pour formuler des milieux.
2.3.6. Utilisation du NaCl et MgSO4 comme stratégie de base pour l'atténuation de
l'effet inhibiteur du savon présent dans le GB
Selon la première stratégie, le NaCl est utilisé pour éliminer le savon par relargage.
Dans le second cas, le MgSO4 sera utilisé pour détruire la propriété tensioactve du
savon en le convertissant en écume. Toutefois, le savon ne sera pas réellement retiré
du GB, de sorte que son rapport C/N restera constant. Les deux approches seront
optimisées pour une meilleure production de H2.
2.3.7. Étude des effets de citrate de fer et de ses nanoparticules vaporisés (spray
dryer) sur la production d'hydrogène par bioconversion du GB
Le citrate de fer solubilisé en utilisant une solution d'eau chaude/NaOH ou formulé
comme des nanoparticules à l'aide d'un "nanospray dryer" sera évalué comme un
supplément de fer et de source de carbone.
2.3.8. Développement d'un système à deux phases pour l'amélioration de la
production d'hydrogène par élimination simultanée des acides organiques du
milieu
Un nouveau système de bioréacteur à deux phases sera développé pour l'extraction
simultanée d'acide organique lors de la production de H2 par bioconversion du GB. À
cet effet, un liquide de phase non aqueuse biocompatible (PNA) sera sélectionné et
testé en bioréacteur à petite échelle (125 mL flacons à sérum). Le système sera
optimisé pour améliorer la production d'hydrogène et l'utilisation maximale du glycérol.
19
2.3.9. Production d'hydrogène en utilisant un bioréacteur de 7,5 L avec une
libération continue du gaz produit et la surveillance en ligne de la production
d'hydrogène par un capteur spécifique au H2
Un procédé semi-continu sera développé où la fermentation sera lancée comme un
processus de traitement par lots à une concentration initiale optimale de GB. Après la
consommation d'une partie du substrat, une solution d’alimentation contenant des
concentrations élevées de GB (par exemple 60-120 g/L) est ajoutée lentement dans le
réacteur de 7,5 L. Pour éviter la perte de biomasse active, un long temps de rétention
hydraulique sera maintenu. Le gaz produit sera périodiquement libéré de l'espace de
tête du réacteur afin de minimiser l'inhibition des réactions.
2.3.10. Évaluation de la capacité de solubilisation du phosphate des rejets
liquides du bioréacteur de production de biohydrogène et son application comme
une alternative au bioengrais utilisé pour solubiliser le phosphate
Les déchets liquides seront recueillis à la fin de la fermentation et ils seront testés
comme agent potentiel de solubilisation du phosphate. Le Ca3(PO4)2, un phosphate
insoluble présent dans le sol est couramment utilisé comme modèle pour déterminer la
capacité de solubilisation du phosphate par les biofertilisants solubilisateurs de
phosphate (PSB). Le déchet est mélangé avec le composé dans des proportions
différentes et la concentration en phosphate soluble est mesurée afin de déterminer
l'efficacité de ces déchets. En cas de succès, un modèle végétal (de plante de soja)
pourrait être utilisé pour vérifier son efficacité comme une alternative potentielle de
PSB.
2.4. Originalité
Basé sur la revue de la littérature ci-dessus l'originalité des travaux prévus pourrait être
décrite comme suit:
a. Aucune recherche n’est disponible sur l'analyse biochimique et élémentaire
détaillée du glycérol brut généré lors de la production du biodiesel à partir des
déchets de viande et des graisses de restaurants.
20
b. Afin de réduire le coût du procédé, le GB doit être évalué en tant que seule
composante pour la production d'hydrogène par fermentation et la possibilité de
recyclage de la biomasse, soit comme inoculum ou comme source de nutriment, doit
être évaluée. De même, afin d'assurer une utilisation maximale de la charge, une
relation possible entre la concentration initiale de GB, la production de H2 et le taux
d’utilisation du glycérol doit être étudiée.
c. Le GB est essentiellement une source de carbone et en le complétant avec les
différentes composantes de milieux synthétiques, la production de H2 pourrait être
améliorée. Cependant, aucune tentative n'a été faite pour évaluer l’efficacité des
différents déchets organiques riches en éléments nutritifs (par exemple boues
d'épuration, les eaux usées d’abattoir, rejets des brasseries).
d. Jusqu'à présent, aucune tentative n'a été faite pour évaluer l'effet interactif des
différentes composantes du GB sur la production de biohydrogène. Dans ce
contexte, la méthodologie de surface de réponse pourrait être adoptée pour évaluer
toutes les interactions possible entre le savon, le méthanol et le NaCl, la production
de H2 et l'utilisation du glycérol.
e. Le savon peut être retiré du GB par relargage à l'aide de NaCl. De même, si le
MgSO4 est ajouté à une solution de GB, l’ion Mg2+ réagit avec les molécules de
savon présentent et détruit les propriétés tensio-actif en les convertissant en écume.
Ainsi, pour se débarrasser de l'effet inhibiteur du savon sur le processus de
bioconversion du GB, ces deux nouvelles stratégies pourraient être appliquées.
f. Le FeCl2 et FeSO4 ont été rapportés comme ayant une meilleure capacité de
production d'hydrogène, car ils peuvent servir comme source de fer. Nous tenons à
proposer le citrate de fer comme une alternative de ces deux composés, car, en plus
d'être une source de fer, il est capable d'agir comme une source de carbone
supplémentaire pour le procédé. De plus, afin d'améliorer sa biodisponibilité (il est
insoluble dans l'eau) le citrate ferrique peut être fourni sous forme de nanoparticules
(nanospraydried).
21
g. Mise au point d’un nouveau système de bioréacteur à deux phases qui pourrait être
développé pour l'élimination in situ des acides organiques et améliorer la production
de H2.
h. Nous avons proposé un procédé de fabrication semi-continu de H2 en réacteur de
7,5 L. En outre, le gaz produit sera périodiquement libéré de l'espace de tête du
réacteur pour éviter toute inhibition due à l'accumulation du H2. Un capteur
d'hydrogène sera testé pour la surveillance en temps réel de la production de H2.
i. Jusqu'à ce jour, la capacité de solubilisation du phosphate à partir des rejets liquides
de la production d'hydrogène par fermentation n'a pas été étudiée. Ainsi, dans la
présente étude les rejets seront utilisés comme solubilisateur de phosphate et en
cas de succès, un modèle d'usine sera utilisé pour déterminer son potentiel comme
une alternative aux biofertilisant servant à solubiliser le phosphate.
Dans l'ensemble, l'originalité de la recherche proposée est, “le développement de
procédés de production de biohydrogène nouveaux et efficaces pour une
meilleure bioconversion du glycérol brut issu de la production de biodiesel avec
l'utilisation durable des déchets secondaires générés au cours du processus”.
22
PARTIE III. SOMMAIRE DES DIFFÉRENTS VOLETS DE RECHERCHE
EFFECTUÉS DANS CETTE ÉTUDE
Un aperçu simplifié de l’ensemble du projet, sous forme de procédé, est illustré à la
Figure 1.1. En outre, des études spécifiques menées dans le cadre de cette thèse ont
été divisées en chapitres suivants :
3.1. Production de biohydrogène: Principes, possibilité et défis (trois articles publiés, un
soumis)
3.2. Production d'hydrogène par la bioconversion du glycérol brut: Une synthèse
pratique (deux articles publiés)
3.3. Effet des impuretés du glycérol brut: spéculations et faits (deux articles publiés)
3.4. Application de la nanotechnologie pour améliorer la bioconversion du glycérol brut
(un article soumis)
3.5. Application potentielle des déchets liquides de fermentation pour la solubilisation du
phosphate (un article publié, un soumis)
3.6. Conception et optimisation de nouveaux bioprocédés pour améliorer la production
d'hydrogène en utilisant le glycérol brut (deux articles soumis)
3.1. Production de biohydrogène: Principes, possibilité et défis (trois articles
publiés, un soumis)
3.1.1. Production microbienne d'hydrogène par bioconversion du glycérol brut:
revue de l’information (chapitre 2, partie I)
Les déchets de procédé de fabrication du biodiesel contenant le glycérol sont un produit
de base ayant un potentiel de production d'hydrogène par fermentation microbienne.
Différents chercheurs ont évalué sa performance en tant que substrat peu coûteux pour
la production d'hydrogène. ces recherches ont montre que potentiel de production est
comparable à tout autre déchet organique actuellement utilisé pour la production
23
d'hydrogène. L'avantage le plus important de l'utilisation du GB sur d'autres substrats
est l’augmentation du bénéfice global des usines de fabrication de biodiesel. Une telle
situation peut encourager la production et l'utilisation des biocarburants, ce qui est
bénéfique pour l'environnement. Cependant, le GB contient de nombreuses impuretés
qui sont des inhibiteurs potentiels de la croissance microbienne.
Les informations
scientifiques sur le prétraitement de GB sont limitées. Ainsi, des efforts additionnels
sont encore nécessaires pour optimiser le prétraitement du glycérol brut pour la
production de biohydrogène. Une méthode d'élimination collective pour les différents
types d'impuretés ainsi qu’une étude de faisabilité de son application à l'échelle
industrielle s’avère essentielles pour l’avenir de la production d'hydrogène à grande
échelle à partir du glycérol brut. Quelques études réalisées en mode continu ont donné
un meilleur rendement de production d’hydrogène que les expériences de traitement
par lots. Ainsi, une recherche plus poussée sur la production d'hydrogène par
fermentation en utilisant le mode continu est recommandée.
Actuellement, le microorganisme le plus étudié pour la production d'hydrogène à partir
du GB brut est Enterobacter aerogenes. D’autres inoculums fréquemment utilisés sont
la microflore indigène mixte contenue dans les déchets organiques.
24
Figure 1. 1: Présentation de la thèse.
25
3.1.2. Enzymes clés: amélioration de la bioconversion du glycérol brut en
biohydrogène (chapitre 2, partie II)
Le métabolisme oxydatif et réducteur est possible pour la bioconversion du glycérol. La
plupart des microorganismes testés pour la production d'hydrogène à partir du GB sont
connus pour avoir des enzymes pour les deux voies. Si le glycérol est principalement
métabolisé par la voie réductrice, la plupart des atomes d'hydrogène présents dans le
glycérol seront utilisés pour générer le 1,3 -propanediol résultant en un rendement de
production d'hydrogène pauvre par mole de glycérol utilisée. Par conséquent, choisir la
bonne souche et optimiser les paramètres du procédé afin de réduire la production de
1,3 -propanediol peut être une stratégie intéressante pour améliorer le rendement de
l'hydrogène à partir du GB.
Une fois le glycérol décomposé en pyruvate, son
métabolisme peut procéder par la voie du Pyruvate formate lyase (PFL) ou Pyruvate
ferredoxine oxido reductase (PFOR). Par contre la voie PFL n'a pas le système
enzymatique nécessaire pour avoir accès au NADH générateur d'hydrogène.
Par
conséquent, le rendement en hydrogène maximale possible pour les microorganismes
ayant une seule voie PFL est seulement de 2 moles de H2 par mole de glucose. De
même, lors de la production d'hydrogène par l'intermédiaire de la voie PFOR ou PFL,
les produits finaux de la fermentation tels que les acides organiques et les solvants sont
produits et la disponibilité des protons pour la production d'hydrogène est limitée.
Cependant, la voie des pentose-phosphates (PPP) est une autre voie efficace qui
permet la conversion de sucres en H2O et CO2 sans la formation de tout autre produit
final de fermentation. Par conséquent, si le métabolisme du glycerol peut être dévié du
métabolisme glycolytique classique pour voie du pentose phosphate (PPP), un
rendement maximal d'hydrogène peut être possible. En outre, des éléments tels que le
Ni, Fe, Mo et S sont essentiels pour la synthèse des différents enzymes
produisant/consommant l’hydrogène (hydrogénases et nitrogénases). Par conséquent,
un bon équilibre de ces éléments dans le milieu de culture est nécessaire pour
améliorer le rendement en hydrogène.
3.1.3. Production de biohydrogène et concept de bioraffinerie: Utilisation
potentielle des déchets liquides de la production d'hydrogène (DLPH) par
fermentation (chapitre 2, partie III)
Au cours de la production d'hydrogène par fermentation sombre, 60-70% du substrat
est transformé en différents sous-produits. L'éthanol, le 1,3 propanediol et l'acide
butyrique sont des sous-produits qui ont une très grande importance commerciale. Si
on considère la production d'hydrogène couplée avec la récupération de l'éthanol en
simultanée; en plus de 1,58 moles de H2, 0,90 mole de l'éthanol pourrait être récupérée
pour chaque mole de glucose transformée. Compte tenu du fait que l'hydrogène est le
principal produit récupéré et que l'éthanol extrait du DLPH n'a pas de coût direct de
production; le rendement est important.
En s’inspirant de cette approche, selon une estimation de bioconversion de 1 kg de
glycérol brut, il est possible de produire 48,14 L de H2 et 307,06 g de 1-3 Propanediol
(PD). Considérant la valeur de marché actuelle de H2 (7,75 à 11,50$/m3) et le PD
(2.43$/Kg- $ 288/L), la quantité de PD qui pourrait être récupérée à partir DLPH a une
importance industrielle. Par bioconversion de 1 kg de glucose, en plus de la production
d'hydrogène de 326,44 L, 299,37 g de butyrate en vrac avec un prix plus élevé que
50$/Kg ou 74,6$/L, peuvent être récupérés comme sous-produit. En variante, le DLPH
est un agent ayant un potentiel de solubilisation du phosphate. En effet, des essais en
laboratoire ont permis d’accroître la concentration en phosphate soluble d'un milieu
aqueux contenant du phosphore inorganique insoluble par un facteur de 36 fois. Par
conséquent, il doit être évalué comme un substitut des biofertilsants solubilisateurs de
phosphore (PSB) commercialement disponible. Comme (i) le PSB est un engrais
biologique avec un succès commercial, (ii) le DLPH n'a pas besoin de traitement en
aval pour cette application, (iii) il n'a pas de coût direct de production et (iv) par rapport
au PSB, il est prévu avoir une durée de vie supérieure. Cette stratégie a un intérêt
considérable en tant que source de revenus supplémentaires pour la production
d'hydrogène par fermentation. Le méthane peut également être produit à partir du
DLPH. Toutefois, cette approche va consommer tous les sous-produits du processus de
production de l'hydrogène qui, autrement, auraient un meilleur marché que le méthane.
Dans l'ensemble, l'approche de la bioraffinerie d'hydrogène porte un potentiel industriel
considérable et, par conséquent, au lieu de se concentrer sur un sous-produit
particulier, l'ensemble du processus doit être évalué comme une stratégie intégrée et
une analyse coûts-avantages appropriée de l'approche est nécessaire.
27
3.1.4. Production de bio-hydrogène par la bioconversion du glycérol brut dérivé
de la production du biodiesel: Une évaluation technico-économique (chapitre 2,
partie IV)
La fermentation en deux étapes, comprenant la fermentation sombre et la photofermentation est une des options les plus prometteuses disponibles pour la production
de biohydrogène. Dans la présente étude, la faisabilité technico-économique d'un tel
processus en deux étapes a été évaluée. L'analyse a été faite sur la base des avancées
récentes dans la production d'hydrogène par fermentation à l'aide de GB comme
matière première. L'étude a été réalisée avec une référence particulière sur le marché
du biodiesel en Amérique du Nord et, plus précisément, les données disponibles pour
les provinces canadiennes et le Québec ont été utilisées. Considérant le matériel, la
puissance, les installations et les coûts d’opération, le coût total associé avec le
procédé a été calculé. De cette analyse, il est ressorti que les composantes de milieux
de culture requis pour le procédé couvrent près de 82 % du coût total de production.
Sur la base de cette analyse, des propositions ont été développées pour la réduction du
coût du procédé.
Également, le procédé a été jugé très bénéfique au point de vue de l'environnement. Il a
été estimé que la bioconversion de 1 kg de GB a le potentiel de réduire de presque 7,66
kg de GES. Aussi, le processus a le potentiel pour atténuer les risques
environnementaux possibles dues à une mauvaise élimination du GB.
3.2. Production d'hydrogène par la bioconversion du glycérol brut: Une synthèse
pratique (deux articles publiés)
3.2.1. Production d'hydrogène à partir du glycérol brut généré lors de la
production de biodiesel à partir de graisses animales et des déchets de
restaurant par fermentation anaérobie et ré-utilisation d’une culture carencée
(chapitre 3, partie I)
Le but de la présente étude était de caractériser le GB afin de déterminer son potentiel
maximal de production de H2, en l'absence de tout nutriment coûteux supplémentaire.
28
En outre, l'utilisation durable des déchets des procédés de production de H2 est l'un des
objectifs. Des différences significatives de la teneur en glycérol, du pH et de la
composition élémentaire ont été observées entre l'huile de soja et le GB généré à partir
d’une usine de biodiesel. Une production maximale de 2022,5 H2 mL/L milieu a été
réalisée par la bioconversion du GB (sans éléments nutritifs supplémentaires) et 10 g/L
de GB a été jugée optimale. De plus, l'addition d’une culture âgée et carencée (50
mg/L) provenant d’une fermentation antérieure dans le procédé permet d’améliorer la
production d’hydrogène de 32,50 %, avec un taux maximum de 1,040 mL/L/jour. De
même, près de 75 % du H2 total a été produit à un pH aussi bas que 3,8, indiquant une
tolérance élevée en acide de la souche utilisée (Enterobacter aerogenes NRRL B407).
3.2.2. Évaluation des différents nutriments supplémentaires pour une meilleure
production de biohydrogène par Enterobacter aerogenes NRRL B 407 à partir du
glycérol brut provenant de déchets de production du biodiesel (chapitre 3, partie II)
La bioconversion du GB, un sous-produit de la production de biodiesel, est un
processus relativement nouveau et prometteur pour la production d'hydrogène.
Cependant, la transformation est fortement limitée par le coût prohibitif attribué aux
composantes de milieux de culture. Dans la présente étude, les déchets, tels que des
BDB (biomasse de déchets de brasserie), RLA (rejets liquides d’abbatoirs), BS (boues
secondaires), MP (marc de pomme), EUIA (Eaux usées de l’industrie de l'amidon) et de
l'urée, une source d'azote moins coûteuse, ont été évalués comme substitut aux
composantes du milieu de culture. L'Espèce E. aerogenes a été utilisée pour l'étude de
cas. Le GB à 10 g/L était la seule source de carbone utilisé et les observations en
laboratoire indiquent que l'addition de RLA, BDB et l'urée peut améliorer la production
de 18,81 ± 3,56, 27,30 ± 3,54 et 38,57 ± 3,66 %, respectivement.
De même, la
production cumulée de 116,41 ± 3,72 mmol H2/L milieu de culture et un taux de 965 ±
35 mL/L de production de H2 maximale/jour ont été obtenus en utilisant l'urée à une
concentration aussi basse que 10 mg/L comme source d'azote supplémentaire. Les
résultats obtenus se sont avérés comparables à ceux provenant d'autres études
disponibles sur la bioconversion du GB, démontrant la pertinence d’utiliser des rejets
29
tels que les BDB, RLA et l'urée comme produit de remplacement pour les composantes
coûteuses du milieu de culture.
3.3. Effet des contaminants du glycérol brut: spéculations et faits (deux articles
publiés)
3.3.1. Étude de l'effet des différentes composantes du glycérol brut sur la
production d'hydrogène par Enterobacter aerogenes NRRL B- 407 (chapitre 4,
partie I)
En utilisant la méthodologie de surface de réponse, le rôle des trois principaux
contaminants du GB (savon, méthanol et NaCl) sur la production d'hydrogène et la
bioconversion du glycérol a été évalué. Parmi ces contaminants, le savon (1 à 3 g/L)
s'est avéré avoir un effet inhibiteur significatif sur la production d'hydrogène (valeur p =
0,0104<0,05). De même, le méthanol (1.3 à 3.3 g/L) a également montré une réponse
similaire dans la plage des concentrations considérées. Fait intéressant, pour tous les
contaminants étudiés, la réponse entre la consommation de glycérol et la production
d’hydrogène n'était pas similaire (comme prévu). La co-utilisation des contaminants
avec le glycérol ainsi qu’un changement métabolique due à la présence de ces
contaminants ont été identifiés comme étant la raison la plus probable pour une telle
observation. Finalement, le NaCl a généré un effet bénéfique à la fois pour la
production de H2 et pour l'utilisation du glycérol. Sur la base de ces observations, une
nouvelle stratégie impliquant du NaCl a été proposé pour l'enlèvement du savon
combiné à l’amélioration de la capacité tampon du milieu à base de GB.
3.3.2. Atténuation de l'effet inhibiteur du savon par traitement au sel de
magnésium du glycérol brut : Une nouvelle approche pour une meilleure
production de biohydrogène à partir des déchets de l'industrie du biodiesel
(chapitre 4, partie II)
Tel que déjà mentionné, le GB contient une série de contaminants et le savon est l'un
d'eux. La teneur en savon de différents échantillons de GB peut varier de 20,5 ± 0,1 à
31,4 ± 0,1 % (p/p). Malheureusement, le savon est un inhibiteur potentiel de la
croissance microbienne. Par conséquent, l'élimination du savon du GB peut avoir un
effet bénéfique sur la croissance microbienne et une production accrue d'hydrogène
30
peut être attendue. Par une stratégie de relargage (salting out), jusqu'à 42 % du savon
a été retiré du GB et l'approche a eu un effet bénéfique sur la production de H2.
Toutefois, l'élimination de plus de 7% de savon a été jugée inhibitrice. En fait, le savon
est utilisé comme co-substrat et, en raison de son enlèvement, le rapport carbone-azote
du milieu peut avoir diminué affectant ainsi la production de biohydrogène. Sans
modifier le rapport carbone-azote du GB, le traitement au MgSO4 peut convertir le
savon en sa forme inactive (écume). Cette approche a permis d’augmenter le taux de
production du H2 (33,82%), la production cumulative de H2 (34,70%) ainsi que
l'utilisation du glycérol (près de 2,5 fois). En outre, le traitement peut augmenter le Mg
(un nutriment) contenu dans le milieu de culture de 0,57 mg/L à 201,92 mg/L.
3.4. Application de la nanotechnologie pour améliorer la bioconversion du
glycérol brut (un article soumis)
3.4.1. Amélioration de la production d'hydrogène par bioconversion des rejets de
l’industrie du biodiesel supplémentés avec du citrate ferrique et de ses
nanoparticules générées par un atomiseur à sec (chapitre 5)
Des nanoparticules de citrate ferrique, de FeSO4, NiCl2 et de Ni-acétate ont été
produites par un atomiseur à sec et évaluées pour leur potentiel d'application en tant
que supplément pour la production d'hydrogène par bioconversion du GB. Parmi ces
suppléments, les nanoparticules de citrate ferrique ont permis d’obtenir un effet
bénéfique sur la production d’hydrogène. Deux types de particules de citrate ferrique
différentes ont été préparés : soit par dissolution dans de l'eau chaude ou dans une
solution de NaOH. Entre ces deux particules, les particules à base de NaOH ont été
inhibitrices pour la production d'hydrogène. Au contraire, les particules à base d'eau
ont amélioré la production d'hydrogène par 31,71 %. En dehors des effets de ces
suppléments, la concentration initiale en substrat a été identifiée comme un facteur
essentiel pour déterminer le rendement en hydrogène. À très faibles concentrations
initiale en GB de 100 mg/L, et en supplémentant la culture avec des nanoparticules de
citrate ferrique, un rendement en hydrogène aussi élevé que 29,9 mol-H2/kg GB a été
atteint. Ce rendement est nettement plus élevé que 8,25 mol-H2/kg GB déjà répertorié
pour la production de biohydrogène par fermentation sombre.
31
3.5. Application potentielle des rejets liquides, riches en acide organique,
provenant du procédé de production du biohydrogène pour la solubilisation du
phosphate insoluble (un article publié, un autre soumis)
3.5.1. Rejets liquides de la production de biohydrogène-une alternative
commercialement intéressante en remplacement des biofertilisants solubilisant le
phosphate (chapitre 6, partie I).
Les rejets liquides issus de la production de biohydrogène sont un excellent agent de
solubilisation du phosphate. De plus, ce rejet a un fort potentiel pour être une alternative
commercialement intéressante aux biofertilisants solubilisant les phosphates. Pour des
essais réalisés en système liquide, une augmentation de la concentration en phosphate
soluble de 23 fois en moins d’une minute et 36 fois après 10 j suivant son application a
été obtenue. Des valeurs supérieures pourraient être obtenues en fonction du procédé
de production d'hydrogène adopté. Contrairement aux biofertilisants traditionnels
solubilisant le phosphate, la solution proposée n'est pas une formulation de
microorganismes vivants et, par conséquent, il devrait avoir une durée de conservation
supérieure. Enfin, le processus de production de biofertilisant proposé pourrait être
intégré au procédé actuel de production du biodiesel afin de mettre à niveau l'industrie
du biodiesel à une branche "profits élevés et zéro déchets".
3.5.2. Solubilisation du phosphate en utilisant les rejets liquides provenant de la
production de biohydrogène (RLPH) et amélioration de l'absorption du phosphore
par les plants de soya (chapitre 6, partie II)
La production cumulative d'hydrogène par le procédé de bioconversion du GB utilisant
Enterobacter aerogenes NRRL B-407 a été établie à près de 0,5 mmol/L milieu de
culture. Une forte diminution de pH du milieu a été observée; ce qui indique que le
début du processus de production d'hydrogène correspond également à une production
d’acides organiques. Le RLPH généré au cours du processus a été testé sur un sol
comme agent de solubilisation du phosphate. Les plants de soya cultivés dans le sol
enrichi avec le RLPH ont montré de 2,18 à 2,74 fois plus d'absorption de phosphore.
L'observation suggère une éventuelle commercialisation du RLPH comme une
alternative aux biofertilisants commerciaux solubilisant le phosphore. Cependant, la
32
période d'application appropriée ainsi que la dose de RLPH doivent être déterminées
par une étude au champ plus complète où les rendements des produits agricoles
pourront être mesurés de façon plus précise.
3.6. Conception et optimisation de nouveaux bioprocédés pour améliorer la
production d'hydrogène en utilisant le GB (deux articles soumis)
3.6.1. Nouveau système anaérobie à deux phases pour la production de
biohydrogène et l'extraction in-situ des acides organiques produits (chapitre 7,
partie I).
Lors de la production d'hydrogène par fermentation sombre anaérobie, l'accumulation
de sous-produits tels les acides organiques favorise l’acidification du milieu de culture
diminuant ainsi les performances du procédé. Pour éliminer ce problème, un nouveau
réacteur en deux phases a été expérimenté afin de produire du biohydrogène et extraire
en simultanée les acides organiques générés lors de la fermentation. En raison de sa
biocompatibilité, l'alcool oléique a été choisi pour l'extraction in situ des acides
organiques. Un rapport de la phase organique:aqueuse de 1: 50 a été jugée optimale
pour le procédé qui permet d'augmenter la production d'hydrogène de 26.6 mmol/L à
29.6 mmol/L-milieu de culture. Pour une fermentation de concentration initiale en GB de
25 g/L, un rendement aussi élevé que 11,7 mmol-H2/L-milieu de culture a été obtenu
pour le système à deux phases.
En comparaison, un système de fermentation
conventionel à une phase génère dans ces conditions 1,48 mmol-H2/L-milieu de culture.
Avec une concentration de substrat initiale élevée de 25 g/L de GB, la concentration
d'acide organique dans le milieu est importante et, par conséquent, l'approche du
réacteur en 2 phases est plus appropriée.
3.6.2. Mise au point d’un bioprocédé en semi-continu à faible coût et suivi en
continu de la production d'hydrogène à partir du glycérol brut (chapitre 7, partie II)
L’acétone, le butanol et l'éthanol ont été détectés dans le GB utilisé comme matière
première pour cette recherche. Les résultats indiquent qu’en raison de la période de
stockage du GB, une fermentation naturelle à partir des microorganismes indigènes
s’amorce et une production d'acétone, butanol et éthanol se produit. Cette étude a
montré que par dilution avec de l'eau distillée, le GB peut être utilisé comme le seul
33
composant du milieu de culture permettant ainsi de concevoir un procédé peu coûteux
pour la production d'hydrogène. Des rendements aussi haut que 5.18 L-H2/L- milieu de
culture ont été observés, ce qui équivaut à 210 mmol-H2/L-milieu de culture. Cette
quantité est nettement supérieure à celle de 2,02 à 2,68 L-H2/L-milieu de culture
connue pour la production d'hydrogène à partir du GB. L'éthanol est le principal sousproduit détecté lors de ces expérimentations. En réduisant la concentration en GB en
passant de 120 g/L à 60 g/L, l'utilisation du glycerol peut être améliorée de 65 % à 91
%.
34
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40
CHAPTER 2
BIOHYDROGEN PRODUCTION: PRINCIPLES, POSSIBILITY AND
CHALLENGES
Chapter 2. Biohydrogen production: principles, possibility and challenges
PART I
MICROBIAL HYDROGEN PRODUCTION BY BIOCONVERSION OF
CRUDE GLYCEROL: A REVIEW
Saurabh Jyoti Sarma1, Satinder Kaur Brar1*, Eduardo Bittencourt Sydney2, Yann
Le Bihan3, Gerardo Buelna3, Carlos Ricardo Soccol2
a
INRS-ETE, Université du Québec, 490, Rue de la Couronne, Québec(QC), Canada
G1K 9A9
b
Bioprocess Engineering and Biotechnology Department, Federal University of Paraná,
Centro Politécnico, Usina Piloto B, CEP 81531-990 Curitiba, Paraná, Brazil
c
Centre de recherche industrielle du Québec (CRIQ), Québec(QC), Canada
(*Corresponding author, Phone: 1 418 654 3116; Fax: 1 418 654 2600; E-mail:
[email protected])
International Journal of Hydrogen Energy (2012) 37(8), 6473-6490.
43
Résumé
L'hydrogène est une source d'énergie propre, sans production de sous-produits nocifs
lors de sa combustion. La bioconversion de différents déchets organiques en
hydrogène est une technologie durable pour la production de bioénergies et a été
étudiée par plusieurs chercheurs. Le glycérol brut généré pendant le procédé de
production de biodiesel peut servir comme matière première pour la production
d'hydrogène en utilisant des procédés par fermentation microbienne. Cet article
présente en revue les recherches réalisées sur le sujet. Une étude actualisée sur la
production mondiale de biodiesel et de glycérine brute et de leur potentiel de marché a
également été réalisée. De même, différentes contraintes techniques de bioconversion
de glycérol brut ont été minutieusement examinées et des stratégies pour améliorer le
rendement de production en hydrogène ont également été proposées. Il a été soulevé
que l'utilisation de glycérol brut provenant de la production du biodiesel afin de produire
de l'hydrogène présente de nombreux avantages par rapport à l'utilisation d'autres
déchets organiques. De façon notable, la bioconversion du glycérol brut en hydrogène a
des avantages économiques directs pour les industries de fabrication de biodiesel.
Cependant, les différentes impuretés présentes dans le glycerol brut sont connues pour
inhiber la croissance microbienne. Par conséquent, le pré-traitement approprié du
glycérol brut est recommandé pour un rendement maximal de production d'hydrogène.
De même, en utilisant le système de bioréacteur approprié et en adoptant un mode de
fonctionnement continu, une recherche plus approfondie sur la production d'hydrogène,
en utilisant du glycérol brut comme substrat, doit être entreprise. En outre, l'isolement
de souches plus productives ainsi que le développement de micro-organismes plus
productif est recommandé. Des stratégies pour l'utilisation de co-culture de microorganismes appropriés comme inoculum pour la bioconversion du glycérol brut et
l’amélioration de la production d'hydrogène ont également été proposées.
Mots clés: biodiesel; bioconversion; glycérol brut; hydrogène; prétraitement
44
Abstract
Hydrogen is a clean source of energy with no harmful byproducts produced during its
combustion. Bioconversion of different organic waste materials to hydrogen is a
sustainable technology for hydrogen production and it has been investigated by several
researchers. Crude glycerol generated during biodiesel manufacturing process can also
be used as a feedstock for hydrogen production using microbial processes. The
possibility of using crude glycerol as a feedstock for biohydrogen production has been
reviewed in this article. A review of recent global biodiesel and crude glycerol production
and their future market potential has also been carried out. Similarly, different technical
constraints of crude glycerol bioconversion have been elaborately discussed and some
strategies for improved hydrogen yield have also been proposed. It has been underlined
that use of crude glycerol from biodiesel processing plants for hydrogen production has
many advantages over the use of other organic wastes as substrate. Most importantly, it
will give direct economic benefit to biodiesel manufacturing industries, which in turn will
help in increasing biofuel production and it will partially replace harmful fossil fuels with
biofuels. However, different impurities present in crude glycerol are known to inhibit
microbial growth. Hence, suitable pre-treatment of crude glycerol is recommended for
maximum hydrogen yield. Similarly, by using suitable bioreactor system and adopting
continuous mode of operation, further investigation of hydrogen production using crude
glycerol as a substrate should be undertaken. Furthermore, isolation of more productive
strains as well as development of engineered microorganism with enhanced hydrogen
production potential is recommended. Strategies for application of co-culture of suitable
microorganisms as inoculum for crude glycerol bioconversion and improved hydrogen
production have also been proposed.
Key words: Biodiesel; Bioconversion; Crude glycerol; Hydrogen; Pretreatment
45
Introduction
Among the natural energy sources, fossil fuels are the major energy sources, which
cover almost 80 % of global energy demand (Ni et al., 2006). However, the storage of
fossil fuels is being rapidly exhausted and additionally combustion of fossil fuels has
encumbered negative impacts on global climate (Aksoy, 2011, Gnana kumar et al.,
2010). Such a situation has triggered a rapid search of alternative energy sources all
over the globe. Bio-fuels being a renewable energy source can be a suitable substitute
of fossil fuels. Biodiesel is one of the mostly investigated bio-fuel, which can be
produced by transesterification of natural fatty acids (Muniyappa et al., 1996). Presently,
in developed nations, numerous plants have been established for commercial biodiesel
production from cheap organic materials. According to Selembo et al. (2009) till 200809, in US alone, there were nearly 176 biodiesel plants producing 9.8 billion liters of
biodiesel per year (Selembo et al., 2009a). During biodiesel production process, a large
volume of glycerol is produced as a byproduct. One volume of glycerol is generated for
every 10 volumes of biodiesel produced (Selembo et al., 2009a, Yazdani et al., 2007).
The concentration of glycerol in biodiesel manufacturing waste may vary from plant to
plant, which may be 1% to 85% (v/v) or higher (Marques et al., 2009, Mu et al., 2006,
Ngo et al., 2011). The crude glycerol produced during biodiesel production generally
contains impurities, such as methanol, soap, salts, oils and solid organic materials (Chi
et al., 2007, Ngo et al., 2011, Selembo et al., 2009b). Glycerol is an important raw
material for food, pharmaceutical and cosmetic manufacturing process (Guerrero-Pérez
et al., 2009). However, due to presence of large amount of impurities; crude glycerol
cannot be directly used to food or pharmaceutical industries and its purification is also
expensive (Escapa et al., 2009). Hence, crude glycerol generated during biodiesel
manufacturing process is of less commercial value (Escapa et al., 2009). Moreover, if
directly released to the environment without proper treatment, it may be hazardous to
the environment (da Silva et al., 2009, Selembo et al., 2009a). Hence, it cannot be
disposed to the environment as a waste byproduct of biodiesel manufacturing process
(da Silva et al., 2009). Alternatively, the glycerol containing waste effluent of biodiesel
manufacturing plant can be used as a cheap substrate for production of different
valuable products, such as 1, 3-propanediol, ethanol, and animal feed, among others
46
(da Silva et al., 2009, Zhengxiang Wang et al., 2001). Production of hydrogen may be
another alternative means for utilization of crude glycerol. Bio-hydrogen production
using crude glycerol form biodiesel manufacturing industry has already been
investigated by various researchers (Ito et al., 2005, Ngo et al., 2011, Selembo et al.,
2009a). It is established that bio-hydrogen has very high energy content and it can be
used as a bio-fuel (Ngo et al., 2011). Heat of combustion for hydrogen is -285.8 KJ/ mol
and
it
has
an
energy
content
of
142.9
KJ/g
(http://www.creative-
chemistry.org.uk/gcse/documents/Module21/N-m21-10.pdf (accessed on 01/06/2014).
Moreover, if hydrogen is used as a fuel, only water is generated as a byproduct
(Selembo et al., 2009a). Hence, considering the increasing concern over global
warming, hydrogen may be considered as the cleanest fuel for future. Therefore,
bioconversion of crude glycerol to hydrogen is an interesting upcoming green field.
In this pursuit, this review presents a systematic and comparative study of presently
available reports on bio-hydrogen production from crude glycerol and tries to find out
the future research that needs to be carried out. Firstly, characteristics of crude glycerol
generated from biodiesel manufacturing processes are reviewed followed by hydrogen
production potential of crude glycerol, various pretreatment methods, bioreactor
systems used for microbial hydrogen production as well as the glycerol bioconversion
potential of different microorganisms. Short comings of crude glycerol bioconversion are
discussed elaborately and various strategies for improved hydrogen production have
been suggested.
Glycerol: by-product of biodiesel production
Transesterification of vegetable oils and animal fats: Glycerol is a byproduct of
biodiesel production process. For biodiesel production, conventionally, triglycerides,
such as vegetable oils and animal fats are mixed with methanol in a reactor. Sodium
hydroxide crystals are added as catalyst of the reaction and the mixture is agitated and
heated to the boiling temperature of methanol (Hak-Joo Kim et al., 2004, Muniyappa et
al., 1996, Schuchardt et al., 1998). When agitation stops, the reaction mixture gets
separated into two phases. The upper layer is methyl ester of fatty acids, which is
biodiesel and the lower layer is crude glycerol containing un-reacted methanol, salt and
47
solid substances present in the raw materials. Figure 2.1.1 represents a schematic
overview of transesterification and biodiesel production process. For every 10 volume of
biodiesel production, one volume of glycerol generated as a byproduct (Selembo et al.,
2009a, Yazdani et al., 2007). Figure 2.1.2 shows the chemical equation for glycerol
production by transesterification of triglycerides (Muniyappa et al., 1996). The crude
glycerol phase generally contains almost 75% glycerol (Muniyappa et al., 1996).
However, glycerol content in biodiesel manufacturing waste may differ for different
manufacturing plants. In an investigation, Ngo et al (2010) have used biodiesel waste
containing 1% (w/v) glycerol for hydrogen production (Ngo et al., 2011). In another
investigation, Selembo et al (2009) have reported the presence of 69.5% (w/v) glycerol
in the waste generated by a biodiesel manufacturing plant (Selembo et al., 2009a).
Similarly, Ito et al (2005) have reported the presence of 41 % (w/v) glycerol in biodiesel
waste collected from biodiesel manufacturing factory, Hiroshima prefecture, Japan (Ito
et al., 2005).
Global biodiesel market and crude glycerol production: Germany, US, France,
Argentina, and Brazil are the top five biodiesel producing nations in the world. Together,
nearly 68.4% of the world’s total biodiesel is produced by these countries
(http://biodiesel-news.com/index.php/2010/03/22/global-biodiesel-market-analysis-andforecasts-to-2020/). According to Global Biodiesel Market (2009 – 2014), a recent
market research report, published by “Markets and Markets”; the total global biodiesel
market is expected to be worth US$12.6 billion by 2014, out of which the European and
American market will share nearly 55.6 % and 28.6 % of the total revenues,
respectively. It is expected that the biodiesel market will grow from $8.6 billion in 2009
to $12.6 billion in 2014 (http://www.marketsandmarkets.com/PressReleases/globalbiodiesel-market.asp). From Figure 2.1.3, it can be seen that Europe is the leading
biodiesel market in the world with a production share of 49.8% which is followed by the
Americas with a production share of 32.8% and Asia Pacific with a share of 4.4% in
2009 (http://biodiesel-news.com/index.php/2010/03/22/global-biodiesel-market-analysisand-forecasts-to-2020/). According to European Biodiesel Board, presently there are
approximately 120 plants in the EU producing up to 6,100,000 tons of biodiesel
annually. These plants are mainly located in Germany, Italy, Austria, France and
48
Sweden (http://www.ebb-eu.org/biodiesel.php). North America is the other front runner
in global biodiesel production. According to a recent survey, in 2010 total biodiesel
consumption in North America was 327 million barrels and during the period of 20062010, biodiesel consumption was increased by a compound annual growth rate (CAGR)
of
23.9%
(http://www.businesswire.com/news/home/20110713006409/en/Research-
Markets-Guide-Biofuel-Consumption-North-America). Similarly, in the same report it has
been estimated that by the end of 2015, total biodiesel consumption revenue of North
America
will
reach
to
$
116.8
billion
with
a
highly
significant
CAGR
(http://www.businesswire.com/news/home/20110713006409/en/Research-MarketsGuide-Biofuel-Consumption-North-America). The United States of America is the
second largest producer of biodiesel in the world which produced 17.7% of global
biodiesel production in 2009 (http://biodiesel-news.com/index.php/2010/03/22/globalbiodiesel-market-analysis-and-forecasts-to-2020/). There were nearly 170 biodiesel
plants in the US by 2008 and soy was mainly used as raw material for biodiesel
production (http://news.cnet.com/8301-11128_3-9930000-54.html). The US market for
biodiesel is expected to reach 6,453 million liters in 2020 (http://biodieselnews.com/index.php/2010/03/22/global-biodiesel-market-analysis-and-forecasts-to2020/). For every ten volume of biodiesel produced nearly one volume of crude glycerol
is generated, hence, a very rapid increase of global crude glycerol production in recent
future can be estimated.
The impurities present in crude glycerol: Due to its high energy content, different
researchers all over the globe have investigated bioconversion of crude glycerol to
valuable products (Ito et al., 2005, Mu et al., 2006, Ngo et al., 2011, Sakai et al., 2007,
Selembo et al., 2009a, Selembo et al., 2009b). They have characterized crude glycerol
from biodiesel manufacturing plants to obtain a broader idea of the impurities present in
it. Chi et al. (2007) reported presence of 16% of impurities in a sample of crude glycerol
collected from a biodiesel refinery. According to them, the impurities mainly present in
the sample were soap, free fatty acids, methanol, unreacted triglycerides, diglycerides,
and monoglycerides (Chi et al., 2007). For a crude glycerol sample collected from a
biodiesel manufacturing plant in Japan; Ito et al. (2005) have reported the presence of
540 g/L total organic carbon (TOC) with ash (8%, w/v), methanol (25%, w/w), 0.04%
49
(w/w) diacylglycerol and 0.01% (w/w) monoacylglycerol (Ito et al., 2005). In another
report by Tang et al. (2009), presence of sodium sulfate 25.8 ± 2.7 % (w/w), methanol
1.5 ± 0.2 % (w/w), water and other compound 5.2 ± 1.0 % (w/w), were confirmed for a
crude glycerol sample (Tang et al., 2009). A crude glycerol sample from biodiesel
manufacturing process may also contain small amount of the oil used for
transesterification (Chi et al., 2007) and any other impurities present in such oil.
Environmental problems of glycerol containing biodiesel waste: As mentioned
above, crude glycerol from biodiesel manufacturing plant may contain up to 25% (w/w)
methanol (Ito et al., 2005) and from different studies it has been concluded that
biodiesel waste containing methanol is inhibitory to microbial growth (Chi et al., 2007,
Tang et al., 2009) and may be toxic to the environment, if released without proper
treatment. Excess methanol present in crude glycerol may contaminate the ground
water. Similarly, due to addition of NaOH in biodiesel manufacturing process, pH of the
generated waste is generally higher and it should be neutralized before waste disposal.
The waste water having high pH, if released without treatment may be harmful to the
biotic community of surrounding environment. Moreover, the waste water with high
salinity and alkalinity may take longer time to be recycled by indigenous microorganisms
of recipient environment which may result in continuous release of offensive odor and
air pollution for longer time.
Different applications of crude glycerol: As mentioned earlier, purification of crude
glycerol is costly (Escapa et al., 2009) and hence its applications in food,
pharmaceutical and personal care industries are not economically significant.
Alternatively, it can be used as a substrate for bioconversion to valuable products.
Different researchers have investigated bioconversion of crude glycerol to numbers of
valuable products and it is summarized in Table 2.1.1. Barbirato et al. (1998) have
reported bioconversion of crude glycerol obtained from colza oil transesterification
process, for production of 1, 3-propanediol. A maximum 0.69 mol 1, 3-propanediol per
mol glycerol was produced. The maximal diol production rate was 1.85 g l-1 h-1, and
butyrate (7.5 g l-1) and acetate (8.1 g l-1) were the only by-products (Barbirato et al.,
1998). Mu et al. (2006) have also reported bioconversion of crude glycerol (85% w/v),
50
collected after lipase-catalyzed transesterification of soybean oil (Mu et al., 2006). Tang
et al. (2009) have reported production of phytase by Pichia pastoris, where crude
glycerol containing methanol from biodiesel processing plant was the sole carbon
source (Tang et al., 2009). Chi et al. (2007) have investigated the production of
docosahexaenoic acid (DHA) by microalgae Schizochytrium limacinum (Chi et al.,
2007). Liu et al. (2011) have described glycolipid production by Ustilago maydis using
crude glycerol as a feedstock (Yanbin Liu et al., 2011). Similarly, Nitayavardhana and
Khanal (2011) have showed that edible fungus Rhizopus microsporus could be grown
using crude glycerol as a carbon source. The resulting biomass contains very high
amount of threonine and can be used as an animal feed supplement (Nitayavardhana et
al., 2011). The above discussion clearly shows that crude glycerol has been
successfully used for different bioconversion processes. However, there is a need to
find out yet another economically attractive and environmentally sound bioconversion
technology for crude glycerol. Production of hydrogen by bioconversion of crude
glycerol may be a suitable option, since, bio-hydrogen has high energy content and it is
a pollution free source of energy, having the potential to be an alternative of increasingly
depleting fossil fuels.
Glycerol: a substrate for bio-hydrogen production
Different substrates presently used for bio-hydrogen: Burning fossil fuel as an
energy source results in global warming (Shaffer, 2009), which is one of the most
serious environmental problems for the world. The burning also causes the acid rain
problem by its combustion gas (Tanisho et al., 1995). In such a situation an alternative
clean energy source is urgently needed. One great advantage of the fermentative
hydrogen production is the broad spectrum of applicable substrates, allowing the
possibility of coupling the energetic utilization of biomass to hydrogen with the
simultaneous treatment of waste materials. Hydrogen produced via bioconversion of
these organic waste materials may be a suitable alternative as combustion of hydrogen
generates only water as byproduct (Selembo et al., 2009a), drastically reducing CO2,
NOx, particulate and other emissions that accompany the use of fossil fuels. Different
plant derived organic materials have been evaluated as feedstock by various
51
researchers for their biohydrogen production potential. Perego et al. (1998) have
evaluated bioconversion of corn starch hydrolysate by Enterobacter aerogenes for its
hydrogen production potential (Perego et al., 1998). Mizuno et al. (2000) have
investigated bean curd manufacturing waste as a substrate for hydrogen production by
anaerobic micro flora (Mizuno et al., 2000). Shin et al. (2003) have reported production
of hydrogen by the mesophilic and thermophilic acidogenic culture using food waste as
a substrate (Shin et al., 2004). Yu et al. (2002) have studied production of hydrogen
using rice winery wastewater as a feedstock. They have reported a maximum yield of
1.9 mol hydrogen per mol substrate used, in an upflow anaerobic reactor by using
mixed anaerobic cultures (Hanqing Yu et al., 2002). Similarly, Tanisho and Ishiwata
(1995) have evaluated molasses for its hydrogen production potential (Tanisho et al.,
1995). Also, numbers of other feedstock were evaluated by different workers for their
hydrogen production potential and they are summarized in Table 2.1.2. From the above
discussion it is clear that a range of cheap and easily available substrates have been
evaluated by various researchers, albeit the yields have been pretty low. At this behest,
there is a need to find an alternative which can give overall yield and lesser mess.
Glycerol as a feedstock for hydrogen production: Glycerol is another feedstock
which can be utilized by many microbes for their growth and has been extensively
studied for its hydrogen production potential. Energy content of pure glycerol is 19.0 MJ/
kg; however, it is 25.30 MJ/kg for crude glycerol which may be due to presence of
methanol and traces of biodiesel (http://www.esru.strath.ac.uk/EandE/Web_sites/0607/Biodiesel/experiment.htm). Such high energy content of crude glycerol indicates its
high potential to be an effective substrate for hydrogen production. Additionally, unlike
most cellulosic waste materials it does not require additional pretreatment to make it
available for the hydrogen producing microorganisms. Moreover, substrates such as
whey and molasses have high demand due to their wide range of application in
industrial fermentations. Further, substrates such as food waste generally contains solid
materials of different origin and need proper grinding and mixing before subjecting it to
fermentation. Hence, for large scale hydrogen production, crude glycerol seems to be
the ideal substrate without having afore mentioned constraints.
52
Kivistö et al. (2010) have demonstrated hydrogen production by Halanaerobium
saccharolyticum utilizing pure glycerol as a substrate (Kivistö et al., 2010). Escapa et al.
(2009) have reported hydrogen production from glycerol by using a microbial fuel cell
(MFC). According to them, if heat-treated anaerobic sludge was used as inoculum, a
maximum volumetric rate of hydrogen production of 0.6 l l -1 d-1 could be achieved
(Escapa et al., 2009). Similarly, Ito et al. (2005) have investigated hydrogen production
from pure glycerol. By using Enterobacter aerogenes HU-101 as inoculum, they have
reported a maximum hydrogen yield rate of 80 mmol l-1 h-1 (Ito et al., 2005). Wu et al.
(2011) also have studied the potential of glycerol as a feedstock for hydrogen
production by Klebsiella sp. HE1 (Wu et al., 2011). Similarly, Seifert et al. (2009) have
evaluated pure glycerol as a substrate for hydrogen production. For their study, a 60
mL glass reactor with working capacity of 30 ml was operated in batch mode and
anaerobic digested sludge was used as inoculum. A maximum 0.41 mol H2 per mol
glycerol was obtained for a medium containing 10 g l-1 glycerol (K Seifert et al., 2009).
Thus, there have been numbers of reports on high level of hydrogen production using
glycerol suggesting its suitability as a feedstock for hydrogen production and they are
summarized in Table 2.1.3. However, pure glycerol is expensive and its use as a
substrate for commercial production of hydrogen will not be economically significant.
Alternatively, crude glycerol produced during biodiesel production may be a good
feedstock
for
microbial
hydrogen
production.
Crude
glycerol
from
biodiesel
manufacturing plant is a waste byproduct of biodiesel refinery and it needs proper
treatment prior to its disposal (da Silva et al., 2009, Selembo et al., 2009a). Meanwhile,
it is a good carbon source and it has the potential to support microbial growth as sole
carbon source. Therefore, it can be used as an alternative feedstock for microbial
hydrogen production. Various investigators have studied the hydrogen production
potential of crude glycerol and reported very high hydrogen yield. Ngo et al. (2010) have
investigated hydrogen production by Thermotoga neapolitana DSM 4359 by using crude
glycerol as a substrate. In a small scale batch experiment using 120-mL serum bottles,
they observed a maximum 0.14 mol-H2 yield per mol-glycerol used (Ngo et al., 2011).
Sakai and Yagishita (2007) have used Enterobacter aerogenes NBRC 12010 to
produce hydrogen from crude glycerol by using bioelectrochemical cells (Sakai et al.,
53
2007). Fernandes et al. (2010) have reported production of hydrogen by anaerobic
sludge using crude glycerol as a substrate (Fernandes et al., 2010). Sabourin-Provost
et al., (2009) have reported hydrogen production using photo-fermentation of pure and
crude glycerol by Rhodopseudomonas palustris. According to them, a maximum of 6
moles of H2 was obtained per mole of glycerol consumed in the process (SabourinProvost et al., 2009). This amount is 75% of theoretical maximum 8 moles H2 production
per mole glycerol (Sabourin-Provost et al., 2009). It indicates that in this case,
intermediate products such as acetic acid, ethanol and butyric acid were further
metabolized to hydrogen, which is otherwise accumulated during dark fermentation.
Chin et al (2003) have also pointed out that intermediate products, such as organic acid
can be further metabolized to CO2 and H2 by photosynthetic organisms (Chin et al.,
2003). Therefore, a two stage fermentation of crude glycerol where dark fermentation
will be followed by photo fermentation by suitable photosynthetic organism should be
considered for further investigation. Further, during bioconversion of crude glycerol,
both H2 and CO2 are produced and CO2 should be removed from the gas mixture to
obtain H2 which can be used as a fuel. In laboratory, NaOH solution is used to remove
CO2 from the biogas mixture and in industrial scale, pressure swing adsorption
technology can be used (Junyapoon et al., 2011, Manish et al., 2008).
From the above discussion based on Table 2.1.2 it is clear that bioconversion of organic
materials for hydrogen production has been extensively studied and feasible. A range of
cheap and waste carbonaceous materials have been investigated as a substrate for
biohydrogen production and good hydrogen yield has been reported for them. Many
workers have used pure glycerol as a feedstock for biohydrogen production reporting
high hydrogen yield potential of glycerol. However, due to higher cost of pure glycerol,
crude glycerol from biodiesel manufacturing process would be a preferred feedstock for
hydrogen production.
Glycerol metabolism and hydrogen production: Both oxidative and reductive
metabolisms of glycerol are known for some species of Klebsiella, Citrobacter,
Clostridium, and Enterobacter (Drożdżyńska et al., 2011). As shown in Figure 2.1.4, in
the oxidative pathway, glycerol is first converted to dihydroxyacetone under catalytic
54
activity of glycerol dehydrogenase. In the next step dihydroxyacetone is phosphorylated
by the glycolytic enzyme dihydroxyacetone kinase. Finally the phosphorylated product is
metabolized through glycolysis (Daniel et al., 1995, Drożdżyńska et al., 2011, Luers et
al., 1997, Macis et al., 1998). In the reducing pathway, glycerol is finally converted to
PD via production of the intermediate product 3-hydroxypropionaldehyde. Conversion of
glycerol
to
3-hydroxypropionaldehyde
is catalyzed
by B12-dependent
glycerol
dehydratase and related diol dehydratases, which is then reduced to PD by the
NADH+H+- dependent enzyme 1,3-propanediol dehydrogenase (Ahrens et al., 1998,
Drożdżyńska et al., 2011, Nemeth et al., 2003).
From Figure 2.1. 4 it can be seen that during oxidative metabolism of glycerol, pyruvate
is formed as an intermediate. According to Drożdżyńska et al (2011) pyruvate formed
during glycerol bioconversion may be metabolized to different end-products by different
organism (Drożdżyńska et al., 2011). According to da Silva et al (2009) ethanol,
butanol, acetone, acetate, butyrate and lactate are some of the possible metabolites of
oxidative metabolism of glycerol (da Silva et al., 2009). In most of the glycerol
bioconversion pathways, along with different metabolites H2 is also produced during
oxidative metabolism. From the stoichiometric equations presented in Figure 2.1.5 it can
be concluded that production of highly reduced end product is accompanied by leaser
H2 yield. Microbial hydrogen production is based on a simple redox reaction: 2H+ + 2e↔
H2 (Mathews et al., 2009). This reaction is catalyzed by some hydrogen-producing
enzymes found in hydrogen producing organisms. Three main such hydrogen producing
enzymes which catalyze most biohydrogen generating reactions are nitrogenases,
[NiFe]- hydrogenases, and [FeFe]-hydrogenases (Mathews et al., 2009, Vignais et al.,
2001). Mathews et al (2009) have illustrated the different metabolic pathways present in
Clostridium acetobutylicum which produce H2 during anaerobic fermentation of glucose
(Mathews et al., 2009). It can be seen from the Figure 2.1.7 that similar to oxidative
metabolism of glycerol, first pyruvate is produced which is then converted to different
metabolites and H2 via different pathways. Pyruvate is broken down to acetyl-CoA via
reduction of a ferredoxin (Fd) catalyzed by pyruvate ferredoxin oxidoreductase.
Reduced ferredoxin (Fd) is then oxidized by a hydrogenase that reproduces oxidized Fd
and hydrogen gas (Mathews et al., 2009, Nath et al., 2004). Some additional amount of
55
hydrogen can be produced during glycolysis when NADH is oxidized by Fd reduction
and NADH-ferredoxin reductase (Mathews et al., 2009, Vardar‐Schara et al., 2008).
Theoretically a maximum of 4 mole H2 can be produced per mole glucose when acetate
is the fermentation end product. However, only 2 mol H2 per mol glucose can be
generated during butyrate production and for reduced end-products such as alcohols
and lactic acid yet lower H2 generation is known (Levin et al., 2004, Mathews et al.,
2009).
Justification of crude glycerol bioconversion to hydrogen: From the above
discussion, it is clear that 1, 3-propanediol (PD) and H2 are the two major products
which can be obtained by bioconversion of crude glycerol. However, due to following
reasons, H2 production is found to be more suitable than PD production by crude
glycerol bioconversion: (a) Glycerol has high hydrogen content (8 numbers) and energy
content
of
hydrogen
is
as
high
as
142.9
KJ/g
(http://www.creative-
chemistry.org.uk/gcse/documents/Module21/N-m21-10.pdf), which is suitable to use it
as a fuel, (b) It will encourage the use of H2 as an alternative fossil fuel which in turn will
reduce greenhouse gas emission, (c) Low yield and low productivity is the major
constraint of bioconversion of glycerol to PD (Zeng et al., 2002). In an investigation,
Abbad-Andaloussi et al. (1998) found that when glycerol alone was used as substrate,
43% of glycerol was oxidized to organic acids to obtain energy for growth and only 57%
was utilized to produce PD (Abbad‐Andaloussi et al., 1998, Saxena et al., 2009).
However, very high bioconversion efficiency has been reported for hydrogen production
using crude glycerol (Sabourin-Provost et al., 2009). (d) Glycerol fermentation to PD is a
two-step reaction catalyzed by Vit-B12-dependent glycerol dehydratase (GDHt) and 1, 3propanediol oxidoreductase (PDOR) enzymes. Therefore, addition of high cost
molecule, Vit-B12 to the medium is required for the production of PD (Laffend et al.,
1997, Saxena et al., 2009). Moreover, when glycerol is used as a substrate, GDHt
undergoes irreversible inactivation by glycerol (Daniel et al., 1995, Corinna Seifert et al.,
2001). e) At the end of bioconversion of crude glycerol to PD, the fermentation broth
generally contains water, residual glycerol, by-products such as acetate, lactate,
ethanol; macromolecules such as proteins, polysaccharides; and unused medium
components. Moreover, PD is known to be hydrophilic in nature and has a high boiling
56
point (Saxena et al., 2009). Because of aforesaid reasons purification of PD from a
fermentation broth is very complex. According to Xiu et al (2008), commonly used
methods for purification of PD are reactive extraction, liquid–liquid extraction,
evaporation, distillation, membrane filtration, pervaporation and ion exchange
chromatography, however, all these methods are known to have numbers of limitations
(Saxena et al., 2009, Xiu et al., 2008). Meanwhile, such constraints are hardly reported
for H2 production as relatively insoluble H2 spontaneously partitions to the gas phase of
a reactor (Sabourin-Provost et al., 2009). Thus, bioconversion of crude glycerol to H2
can be considered as the most suitable option for utilization of biodiesel industry derived
crude glycerol.
Challenges to be addressed: It is clear that akin to other cheap organic substrates,
crude glycerol from biodiesel manufacturing process may also be a potential feedstock
for industrial production of hydrogen using microbial process. However, to convert it to
be a reliable and economically significant option for microbial hydrogen production, the
following issues should be addressed successfully:
(a) From Figure2.1. 4 it can be concluded that in many microorganism there exist
two biochemical pathways for glycerol metabolism. H2 gas is generated during
bioconversion of glycerol through oxidative pathway; however, PD is produced during
reductive metabolism. When both the pathways exist in the same organism, some
amount of H2 is consumed for PD production. According to Selembo et al. (2009),
during anaerobic fermentation of glycerol, for production of 1 mol of PD, 1 mol of H2 is
consumed (Selembo et al., 2009a). Therefore, final yield of H2 is reduced by directly
consuming the produced H2 for simultaneous production of PD and also by making most
of the substrate unavailable for H2 production by reducing it to PD. Continuous removal
of produced hydrogen from fermentation broth by pursing N2 may result in improved
hydrogen production by making it unavailable for subsequent PD production. Similarly,
genetic engineering strategies for blocking PD production pathway in H2 producing
organism may also be a viable option for improved H2 production. Another alternative
possibility is to further break down of PD produced during glycerol metabolism to H 2 by
developing novel strategies. These strategies may involve chemical treatment of PD,
57
produced during anaerobic fermentation of crude glycerol, to ensure its availability as a
substrate for hydrogen production. Otherwise, suitable metabolic engineering strategies
may also be devised for the same purpose.
(b) Usually, heat pretreatment is used for hydrogen producing inocula to inhibit
hydrogen consuming organisms, such as methanogens without reducing the activity of
hydrogen producing organism (Ginkel et al., 2001, Selembo et al., 2009a). Other
methods for pretreatment of hydrogen producing inocula are ultra-sonication,
acidification, sterilization, and freezing/thawing (Cai et al., 2004, Jan et al., 2007,
Selembo et al., 2009a, CC Wang et al., 2003). However, according to Selembo et al.
(2009) efficiency and effect of these different pretreatment methods on hydrogen
production and glycerol fermentation is not well documented (Selembo et al., 2009a).
Therefore, all such methods need to be verified for glycerol fermentation and hydrogen
production efficiency for better hydrogen yield.
(c) Accumulation of fermentation end product such as acetic acid, butyric acid
resulted in lower yield of hydrogen during dark fermentation (Mathews et al., 2009, Van
Ginkel et al., 2005). Undissociated acid concentration of 19 mM in the fermentation
broth is enough to initiate solventogenesis and cease of hydrogen production (Mathews
et al., 2009, Van Ginkel et al., 2005). Therefore, further conversion of end products of
glycerol fermentation to hydrogen is necessary for maximum hydrogen yield. Two stage
fermentation of glycerol where dark fermentation is followed by photo fermentation or
microbial fuel cell technology to produce hydrogen may be a suitable option. Other
alternative is to use membrane bioreactors in continuous processes, allowing biomass
to be retained and fluid (containing volatile fatty acids) to be washed out (Oh et al.,
2004). Alternatively, co-culture of hydrogen producing organism and microbes that can
produce hydrogen from glycerol fermentation end products may be another option.
(d) It can be seen from the Figure 2.1. 5 that high hydrogen yield is possible when
acetic acid is produced as fermentation end product instead of butyric acid and other
solvents. Morimoto et al (2005) have reported that in C. paraputrificum M-21 improved
hydrogen yield has been achieved by over-expression of hydA gene which encodes
[FeFe]- hydrogenase (Mathews et al., 2009, Morimoto et al., 2005). Almost 1.7-fold
increase in hydrogen production over wild strain was accompanied by increased acetic
58
acid production due to over-oxidation of NADH (Mathews et al., 2009, Morimoto et al.,
2005). Other similar strategies should be developed for increased acetic acid production
during glycerol fermentation for improved hydrogen yield.
(e) From different reports, it can be found that methanol and soap, the major
impurities present in crude glycerol are inhibitory towards microbial growth (Chi et al.,
2007, Ngo et al., 2011). Therefore for improved hydrogen production by bioconversion
of crude glycerol suitable pretreatment technology for biodiesel industry derived glycerol
should be developed. Such technology should be suitable for scaling up to industrial
scale and economically sound.
(f) Round the year availability of crude glycerol should be ensured for large scale
production and use of hydrogen as a fuel. Government agencies should take the
initiative for popularization of use of hydrogen energy as well as to develop proper
market for biohydrogen to ensure economic viability of large scale bioconversion of
crude glycerol for hydrogen production.
Pretreatment of crude glycerol
Importance of crude glycerol pretreatment: As it is mentioned earlier, the
transesterification process for biodiesel production involves mixing of vegetable oil and
animal fats with methanol and addition of sodium hydroxide as catalyst (Hak-Joo Kim et
al., 2004, Muniyappa et al., 1996, Schuchardt et al., 1998). Therefore, apart from
glycerol, a typical biodiesel waste may contain high concentrations of salts (e.g., sodium
chloride), methanol, and precipitated solids (Vicente et al., 2004, Yazdani et al., 2007),
which may affect the microbial growth, when added as a substrate for bioconversion
(Chi et al., 2007, Ngo et al., 2011). Ngo et al., (2010) have reported that when crude
glycerol was directly mixed with culture medium for bioconversion purpose, soaps
precipitated from the liquid media, which was found detrimental to cell growth (Chi et al.,
2007, Ngo et al., 2011). Athalye et al. (2009) have reported the negative effect of crude
glycerol containing methanol and soap on production of eicosapentaenoic acid by
Pythium irregular (Athalye et al., 2009). Similarly, Pyle et al. (2008) have also reported
the inhibitory effect of crude glycerol containing methanol and soap on production of
docosahexaenoic acid by Schizochytrium limacinum (Pyle et al., 2008). In order to avoid
59
such incidences, crude glycerol should be pre-treated before using as a feedstock for
hydrogen production. To investigate the effect of pretreatment of crude glycerol on
hydrogen production, Ngo et al. (2010) had conducted a study where pre-treated
glycerol and untreated glycerol waste were used as the substrate for T. neapolitana.
According to the authors, the level of H2 produced from pre-treated glycerol waste, from
where alcoholic compounds are removed by rotary evaporation and solid substances
were removed by centrifugation, was much higher than that from glycerol waste without
treatment (Ngo et al., 2011).
Different methods of crude glycerol pretreatment: As discussed earlier, soap and
methanol are the major impurities present in crude glycerol and they are inhibitory to
microbial growth (Athalye et al., 2009, Chi et al., 2007). To use the crude glycerol as a
feedstock for bioconversion, methanol can be removed by autoclaving (Athalye et al.,
2009, Ethier et al., 2011). According to Denver et al (2008) methanol evaporates at 65
°C, hence the autoclave process lasting for 15 min at 121 °C is capable of removing
significant amount of methanol from crude glycerol (Pyle et al., 2008). Alternatively,
since boiling point of methanol is only 65°C which is far lower than that of water and
glycerol; methanol removal by controlled distillation of crude glycerol can also be
considered. A very simple distillation set up will be required for such a process and it will
be easy to scale up for industrial application. In a recent investigation on hydrogen
production from crude glycerol, Ngo et al. (2010) have reported the removal of methanol
and ethanol by rotary evaporation at 45oC, where solids were precipitated by
centrifugation at 15,000 rpm for 15 min (Ngo et al., 2011).
Soap is the other major impurity present in crude glycerol, which can be removed by
precipitation from the liquid medium through pH adjustment (Athalye et al., 2009, Ethier
et al., 2011). Chi et al. (2007) have used the following procedure to remove soap from
crude glycerol: (i) crude glycerol was first mixed with distilled water at a ratio of 1:4 (v/v)
to reduce the viscosity; (ii) pH of the solution was adjusted to 6.5 with hydrochloric acid
to convert the soluble soap into insoluble free fatty acids; (iv) finally precipitated fatty
acid was removed from the crude glycerol solution by centrifugation at 5000 rpm (Chi et
al., 2007). The main advantage of this method is that the final pH of resulting solution
60
(6.5) is suitable for H2 production by wide range of microorganisms. Similarly, Ethier et
al. (2011) have adopted the following procedure: (i) crude glycerol was mixed with
distilled water at a ratio of 1:4 (v/v) to reduce the viscosity of the solution, (ii) soap was
precipitated by converting it to insoluble free fatty acid by adjusting the pH of the
solution to 3 by using sulfuric acid, (iii) then the fatty acid-precipitated solution was kept
static for 30 min to allow free fatty acid and glycerol to separate into two phases, (iv)
finally, the upper free fatty acid phase was removed from the crude glycerol phase
through a separation funnel (Ethier et al., 2011). Sodium chloride is another major
impurity present in crude glycerol from biodiesel production process (Vicente et al.,
2004, Yazdani et al., 2007). Chiu et al. (2006) have evaluated a method for removal of
sodium from glycerol solutions by crystallization/precipitation of hydroxyapatite (HAP)
through the co-addition of lime [Ca (OH) 2] and phosphoric acid (Chiu et al., 2006).
However, presence of sodium chloride in crude glycerol also offers a unique opportunity
to use salt resistant H2 producing microorganisms for crude glycerol bioconversion
without
having
much
competition
for
substrate
utilization.
Therefore,
before
recommending any method for sodium chloride removal, the possible advantage offered
by its presence should also be considered.
Thus, various methods have been evaluated for removal of major impurities from crude
glycerol. However, no attempt has been made to scale up any of above mentioned
process for impurity removal from crude glycerol. Therefore, information on suitability of
such method for application in large scale bioconversion of crude glycerol is lacking.
Hence, detailed study on optimization of such pretreatment methods, investigation of
effect of such pretreatments on bioconversion of crude glycerol and their economic
feasibility for industrial application should be evaluated. Moreover, the possibility of a
collective removal method for all the major impurities present in crude glycerol should
also be explored.
Bioreactor systems for hydrogen production
Different types of reactors evaluated for bio-hydrogen production: To evaluate
hydrogen production using different organic waste materials including crude glycerol
from biodiesel manufacturing plant, a range of reactor types have been used. A
61
bioreactor used for this purpose may be a small serum vial of 10 mL working volume or
a 2.5-L fermentor. Kivistö et al. (2010) have reported the use of airtight glass tube of 10
mL working volume for hydrogen production by using Halanaerobium saccharolyticum.
It has been reported that by using pure glycerol as a substrate, nearly 1.61 mol-H2 could
be generated from each mol of glycerol, where the study was carried out in batch mode
(Kivistö et al., 2010). Fernandes et al. (2010) have studied hydrogen production from
crude glycerol by using a 2-L glass flasks consisting of 1-L of liquid volume and 1-L of
headspace. The flask was inoculated with the inoculum derived from a packed-bed
reactor used to produce hydrogen from a sucrose-based synthetic substrate and
anaerobic environment was maintained using N2 (Fernandes et al., 2010). Ito et al.
(2004) have described a packed-bed reactor of 60 mL working volume which was
operated in continuous mode. Crude glycerol containing fresh medium was supplied
from the bottom, and the resulting effluent was discharged from the top of the reactor,
with the help of a peristaltic pump (Ito et al., 2005). In another study, Yu et al. (2002)
reported hydrogen production from rice winery wastewater using a 3.0-L up-flow
reactor. The Plexiglas made reactor was of 90 mm diameter (internal) and a height of
480 mm. Using mixed anaerobic culture, under continuous operation of the reactor, a
maximum 2.14 mol-H2 per mol hexose was reported by the authors (Hanqing Yu et al.,
2002). Use of bio-electrochemical cell is another widely investigated method for
hydrogen production. Different workers have evaluated the use of pure or crude glycerol
as a substrate for hydrogen production using bio-electrochemical cells (Escapa et al.,
2009, Sakai et al., 2007, Selembo et al., 2009b). Sakai and Yagishita (2007) have
described a bio-electrochemical two-compartment reactor with a cation-exchange
membrane separating the compartments and crude glycerol was used as a feedstock
for hydrogen production by Enterobacter aerogenes NBRC 12010 (Sakai et al., 2007).
Figure 2.1.6 represents a microbial electrolysis cell used for H2 production by
bioconversion of glycerol. The basic principle of the system is that during bioconversion
of glycerol, electron is donated to the anode, and with the help of an external power
supply, free protons are reduced on the cathode to produce H2 (Escapa et al., 2009,
Hongqiang Hu, 2009, Hong Liu et al., 2005, Logan et al., 2008).
62
From the above discussion (Table 2.1.4), it is clear that a range of bioreactor setup has
been evaluated for hydrogen production from cheap organic materials including crude
glycerol from biodiesel industry. They may be simple serum bottles of different size
(Akutsu et al., 2009, Kivistö et al., 2010, Marques et al., 2009, Sabourin-Provost et al.,
2009, Selembo et al., 2009a), laboratory scale fermentor with agitation and pH control
(Wu et al., 2011, Yokoyama et al., 2007a), pack-bed or up flow reactor constructed from
Plexiglas (Ito et al., 2005, Hanqing Yu et al., 2002), or may be bio-electrochemical cells
(Escapa et al., 2009, Sakai et al., 2007, Selembo et al., 2009b). However, based on the
hydrogen yield potential reported, a particular setup could not be considered superior to
the rest. It is also observed that most of the experiments were carried out in batch mode
using serum vials (Akutsu et al., 2009, Kivistö et al., 2010, Ngo et al., 2011), as the
studies are still in their infancy stage. In a report on hydrogen production from biodiesel
processing waste, Ito et al. (2004) have indicated that the biodiesel wastes containing
glycerol should be diluted with a synthetic medium to increase the rate of glycerol
utilization. They have reported that the production of H2 decreased with an increase in
the concentrations of biodiesel wastes (Ito et al., 2005). Therefore, for bioconversion of
large volume of diluted crude glycerol, a continuous system may be more suitable than
a batch system, once it assures maximum mixing and homogeneity and controlled
concentration of volatile fatty acids (based on organic loading rate and hydraulic
retention time), on which relatively less reports are available. Further, unlike mostly
reported free cell systems for crude glycerol bioconversion, suitable reactor with
immobilized cell system, such as packed bed and membrane reactors may offer dual
benefits of higher initial cell density and protection of microorganisms from impurities
present in crude glycerol.
Important process parameters and their optimum levels: From Table 2.1.4, it is
clear that microbial hydrogen production has been investigated using different reactor
types and chosen optimum process parameters have been different for separate cases.
Ngo et al. (2010) have investigated hydrogen production potential of Thermotoga
neapolitana DSM 4359 using biodiesel manufacturing waste as feedstock. They have
reported high hydrogen yield by the hyperthermophilic eubacterium, where the
experiment was carried out at 75ºC and pH 7.5 (Ngo et al., 2011). Selembo et al. (2009)
63
have investigated the hydrogen production by anaerobic fermentation of crude glycerol
with heat-treated mixed cultures. They have carried out the experiment at 30º C and pH
6.2 (Selembo et al., 2009a). Perego et al. (1998) have studied hydrogen production by
E. aerogense, using corn starch hydrolysate as a substrate. The reactor was operated
at 40±0.5 ºC and pH 5.5 and a high yield of 1.8 mmol H2 per mmol glucose was
reported by the authors (Perego et al., 1998). From the above discussion, it is clear that
the temperature range used by various researchers for hydrogen production varied from
25 º C to 75 º C (Escapa et al., 2009, Ngo et al., 2011), and similarly pH 4.5 to 7.5 was
reported (Fang et al., 2006, Ngo et al., 2011). The process parameters may vary
according to the mode of operation or the biological agent (microorganism) used in the
process. Therefore, a particular temperature or pH cannot be considered optimum for
hydrogen production process as a whole. However, when the process is carried out
using a particular strain, there will be an optimum level for such parameters.
Environmental conditions and media composition when working with industrial complex
wastewaters plays an important role in process development because it influences
drastically hydrogen production. Many studies reported the influence of Zn, Cu,
nitrogen, phosphorous, magnesium and iron in dark fermentation processes (Chong et
al., 2009, Oztekin et al., 2008, Winter, 2005, Han Qing Yu et al., 2001). Hawkes et al.
(2007) reviewed the media composition for hydrogen production and found that apart
from N and P source, only K, Mg and Fe are common in all recipes analyzed so far
(Hawkes et al., 2007). More details of microelements consumption and dark
fermentation evolution might be evaluated but it is clear that by knowing media
composition supplementation of micronutrients and/or dilution with water can be
predicted (saving time and money).
Microorganisms used for glycerol bioconversion
Microorganisms presently used for glycerol bioconversion: As glycerol has been
used for production of different products; various microorganisms have been evaluated
for their glycerol bioconversion ability. Ngo et al. (2010) reported glycerol bioconversion
and
hydrogen
production
using
Thermotoga
neapolitana
DSM
4359,
a
hyperthermophilic eubacterium. They have reported nearly 2.73 mol-H2 production for
64
each mol glycerol used (Ngo et al., 2011). Selembo et al. (2009) have investigated
bioconversion of glycerol by heat treated mixed microbial culture. They have reported a
maximum production of 0.31 mol H2 per mol-glycerol consumed as well as CO2 and PD
as byproduct (Selembo et al., 2009a). Barbirato et al. (1998) have used Klebsiella
pneumoniae ATCC 25955 to produce PD by bioconversion of glycerol (Barbirato et al.,
1998). Similarly, Tang et al., (2009) used Pichia pastoris to produce Phytase by using
crude glycerol as a substrate (Tang et al., 2009). Nitayavardhanaa et al (2011) have
recently reported the possibility of production of animal feed supplement from crude
glycerol by using edible fungus, Rhizopus microspores (Nitayavardhana et al., 2011).
At high temperature, hydrogen production is more exergonic, extreme- and hyperthermophiles show resistance to high hydrogen partial pressures (van Niel et al., 2003),
which otherwise would cause a metabolic shift to production of more reduced products.
One advantage of fermentation at extreme temperatures is that the process is less
sensitive to contaminations but on the other hand it is complicated to achieve a positive
economic relation between the energy used to heat and maintain the reactor at high
temperatures and the H2 production. Moreover, extreme thermophilic anaerobic bacteria
usually grow low densities resulting in low production rates. Researchers have
investigated the bioconversion of glycerol using monoculture or mixed microbial
consortia. As it is shown in Table 2.1.5, Enterobacter aerogenes is the most studied
organism for hydrogen production by using crude glycerol. Mixed microbial culture from
environmental sources is the other mostly used inoculums for glycerol bioconversion;
however, some constraints of process stability do exist and might be considered
especially when industrial wastewaters with continuous composition variation are used.
Phowan et al. (2010) have demonstrated that co-culture of C. butyricum TISTR 1032
and E. aerogenes TISTR 1468 was capable of reducing the lag phase of hydrogen
production from cassava pulp hydrolysate with the maximum cumulative hydrogen
production of 458 ml. They have reported that production of H2 was approximately
14.50% higher than that of using only C. butyricum TISTR 1032 alone (Phowan et al.,
2010). Similarly, Wang et al. (2008) have demonstrated a co-culture of Clostridium
acetobutylicum X9 and Ethanoligenens harbinense B49 capable of producing 1810 ml
H2/L-medium using microcrystalline cellulose as substrate (Aijie Wang et al., 2008).
65
However, the use of co culture of two suitable strains for bioconversion of crude glycerol
to hydrogen has not been explored completely.
Use of co-culture for glycerol bioconversion to hydrogen may play a significant role in
improving hydrogen yield. It is observed that during glycerol metabolism along with
hydrogen numbers of fermentation end products such as acetic acid, butyric acid,
ethanol and butanol may be produced (Figure 2.1. 7). However, accumulation of such
products in the fermentation broth is known to be inhibitory towards hydrogen
production (Mathews et al., 2009). Li et al (2007) have shown that intermediate products
of glycerol fermentation such as butyric acid and ethanol can be further converted to
hydrogen by acetogenic bacteria (Li et al., 2007). Therefore, co-culture of acetogenic
bacteria along with hydrogen producing organism for glycerol bioconversion may reduce
the accumulation of metabolites and improve hydrogen yield. However, acetogenic
strain to be used for glycerol bioconversion as co-culture should be verified for
preferential metabolism of fermentation end products in presence of glycerol as major
substrate.
Methanol and soap are the two major impurities present in crude glycerol and they are
known to have inhibitory effect on microbial growth (Chi et al., 2007, Ngo et al., 2011).
Bacteria, such as Sporomusa acidovorans is known to have methanol degradation
potential (Cord-Ruwisch et al., 1986). Similarly, some species of Klebsiella, Escherichia,
Enterobacter are known to have soap degradation potential (Amund et al., 1997).
Therefore, a co-culture of hydrogen producing bacteria and methanol or soap degrading
bacteria may be helpful for improve bioconversion of crude glycerol to hydrogen.
However, soap or methanol degradation efficiency of proposed co-culture should be
verified in presence of crude glycerol as alternative substrate, before using it for glycerol
bioconversion.
Similarly, PD is another product which may be produced during glycerol bioconversion
to
hydrogen.
Therefore,
a
co-culture
of
suitable
microorganism which
can
simultaneously utilize PD or intermediate product of PD pathway and can produce
hydrogen via glycerol fermentation may be a suitable option of crude glycerol
bioconversion.
66
Genetic and metabolic engineering for improved hydrogen production: Genetic
and metabolic engineering may be seen as potential tools for improvement of hydrogen
production by bioconversion of crude glycerol. In recent years, various researchers
have investigated microbial hydrogen production by using engineered microorganisms
and have reported increased hydrogen yield (Chittibabu et al., 2006, Eui-Jin Kim et al.,
2008, Maeda et al., 2007, Morimoto et al., 2005). Kim et al. (2008) have reported
hydrogen production by recombinant Rhodobacter sphaeroides containing the genes for
pyruvate metabolism of Rhodospirillum rubrum. They have observed that under
photoheterotrophic conditions, H2 production by the recombinant Rhodobacter
sphaeroides increased up to two fold and produced 4 mol H2 per mol of glucose
compared to 2 mol H2 per mol of glucose by the strain without the gene from R. rubrum
(Eui-Jin Kim et al., 2008). Similarly, Maeda et al (2007) have reported a recombinant E.
coli strain expressing the bidirectional [NiFe]-hydrogenase from Synechocystis sp. PCC
6803. A 41 fold increase in hydrogen production over the wild type has been reported
for the recombinant strain. According to the authors, by using an optimized medium, the
recombinant E. coli cells are also capable of producing 2 folds higher hydrogen than the
well-studied Enterobacter aerogenes HU-101 (Maeda et al., 2007). Chittibabu et al.
(2006) also have developed a genetically modified strain of E. coli containing [FeFe]hydrogenase gene from Enterobacter cloacae IIT BT-08. 3.12 mol-H2 yield per mol
glucose has been achieved for the genetically modified strain, which was much higher
than its wild type (Chittibabu et al., 2006).
A number of successful genetic engineering attempts have been made for improvement
of microbial hydrogen production. However, hydrogen production potential of a
recombinant strain by using different organic wastes materials as feedstock is not yet
documented. In a recent review, Mathews et al (2009) suggested that in order to
increase the economic viability of hydrogen production, various substrate utilization
pathways should be engineered into hydrogen-producing organisms (Mathews et al.,
2009). Therefore, a genetically engineered microbial strain capable of efficient
bioconversion of crude glycerol may improve the economic viability of microbial
67
hydrogen production. Recently, by using adaptive evolution and chemical mutagenesis
followed by growth experiment on glycerol, Hu et al (2009) have developed an improved
Escherichia coli strain (HW2) that produces 20-fold more hydrogen in glycerol
containing medium (0.68 ± 0.16 mmol-H2 l-1 h-1) (Hongbo Hu et al., 2010). Yazdani et al
(2007) also have described a recombinant Escherichia coli strain capable of
bioconversion of glycerol to ethanol and hydrogen (Yazdani et al., 2007).
Conclusion and future perspectives
Hydrogen can be considered as the cleanest alternative of fossil fuels as no harmful
byproduct generated during its combustion and it can be produced from renewable
energy sources. Glycerol containing waste from biodiesel manufacturing process is a
potential feedstock for microbial hydrogen production. Different researchers have
evaluated its performance as a cheap substrate for hydrogen production and indicated
that its hydrogen production potential is comparable to any other organic waste
presently used for hydrogen production. The most important advantage of using crude
glycerol over other substrates for hydrogen production is that it will increase the overall
profit of biodiesel manufacturing plants. Such a situation may encourage the production
and utilization of biofuels, which is environmentally beneficial. Biohydrogen can be
burned for heat generation and/or converted to electricity through fuel cells and be used
in the biodiesel process, replacing and avoiding dependence of other non green energy
sources. However, crude glycerol contains many impurities which are inhibitory to
microbial growth and hydrogen production. Scarce literature reports are available on
pretreatment of crude glycerol used for hydrogen production. Hence, further
investigation is still required to optimize crude glycerol pretreatment for biohydrogen
production. A collective removal method for different types of impurities and feasibility
study of its industrial scale application may be helpful for crude glycerol bioconversion
and large scale hydrogen production in future. Accumulation of fermentation end
products, such as acetic acid and butyric acid is known to have negative effect on
overall hydrogen yield. Hence, alternative strategy, such as further conversion of
fermentation end product into CO2 and H2 by photo fermentation should be investigated
in detail. It should be noted that maximum production of 6 mole H2 per mole glycerol
68
has been achieved by photo fermentation which is 75% of theoretical maximum 8 mole
H2 per mole glycerol.
Similarly, most investigations on crude glycerol bioconversion have been carried out in
serum bottle scale batch reactors. Only, a few studies carried out in continuous mode
have given better yield of hydrogen than batch experiments. Hence, further investigation
of microbial hydrogen production using continuous mode is recommended. The most
investigated microorganism for hydrogen production from crude glycerol is Enterobacter
aerogenes. Other frequently used inoculum is indigenous mixed micro-flora of organic
waste materials. It is a common practice that when a microbial consortium is used as
inoculum, pretreatment of such inoculum is made to eliminate hydrogen consuming
organisms, such as methanogens. Ultrasonic, thermal and acid treatments are some
common method for inoculum pretreatment. However, their role on overall hydrogen
production is not well documented. Hence, detailed study and optimization of such
methods may play a vital role large scale hydrogen production in future. Alternatively,
co-culture of two different strains can also be evaluated for crude glycerol
bioconversion. Application of a co-culture, which is capable of reducing the
accumulation of fermentation end products by simultaneously metabolizing it to H2, is an
interesting subject for future investigation. Further, genetically engineered microbial
strains may enhance the hydrogen production potential of crude glycerol.
Abbreviations
PD= 1, 3-propanediol, EU= European Union, GWh= gigawatt hour, TOC= total organic
carbon, DHA= docosahexaenoic acid, MFC= microbial fuel cell, COD= chemical oxygen
demand, mM= millimolar, HAP= hydroxyapatite, GDHt= glycerol dehydratase, PDOR=
1, 3-propanediol oxidoreductase
Acknowledgments
The authors are sincerely thankful to the Natural Sciences and Engineering Research
Council of Canada (Discovery Grant 355254), Le Centre de recherche industrielle du
Québec (CRIQ), MAPAQ (No. 809051) and Ministère des Relations internationales du
69
Québec (coopération Paraná-Québec 2010-2011) for financial support. The views or
opinions expressed in this article are those of the authors.
70
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81
Table 2.1. 1: Different valuable products obtained from crude glycerol generated during bio-diesel production
Source of bio-diesel production
Glycerol
Impurities
waste containing glycerol
content
glycerol
present
in
crude
Maximum product yield using
Ref.
crude glycerol as feedstock
(w/w)
M Energy Co., Pyeongteak city,
1%
South Korea.
Biodiesel
manufacturing
factory,
41%
Hiroshima prefecture,
Methanol
and/ethanol;
solid
2.73±0.14 mol-H2 mol
substances
consumed
Total organic carbon (TOC) 540 g/l,
63 mmol-H2.l .h
ash (8%, w/v), methanol (25%,
-1
-1
-1
glycerol
and 0.85 mol-
(Ngo et al., 2011)
(Ito et al., 2005)
-1
ethanol.mol glycerol consumed.
w/w), 0.04% (w/w) diacylglycerol
Japan
and 0.01% (w/w) monoacylglycerol.
BDF plant, Ishikawa Prefecture,
__
0.77
Japan
Nittany Biodiesel, State College, PA
mol-H2
.mol-1
glycerol
(Sakai et al., 2007)
consumed
69.5%
Chemical oxygen demand (COD) of
0.31 mol-H2 .mol-1 glycerol and
1,300 ± 100 mg/l.
0.59 mol-1, 3-propanediol (PD).
(Selembo et al., 2009a)
mol-1 glycerol consumed
Nittany Biodiesel, State College, PA
69.5%
Methanol,
sodium
hydroxide,
sodium methylate and a chemical
oxygen demand (COD) of 1160 ±
100 mg/l
82
0.41 ± 0.1 m3 H2. m-3 d-1
(Selembo et al., 2009b)
Glycerol
Impurities
present
in
crude
content
glycerol
__
0.69
Ref.
(w/w)
ROBBE, Compie`gne, France
80%
mol-PD.
mol
-1
glycerol
consumed and butyrate 7.5 g-l
(Barbirato et al., 1998)
-1
-1
and acetate 8.1 g-l were the byproducts
Prepared in laboratory by using
85%
__
1, 3-propanediol 53 g-l
transesterification of soybean oil
–1
crude
(Mu et al., 2006)
U-ml-1
(Tang et al., 2009)
glycerol.
Integrity Biofuels, Indiana, USA
67.5 ± 3.2
Sodium sulfate 25.8 ± 2.7 %,
Phytase
(1125
%
methanol 1.5 ± 0.2 %, water and
supernatant)
other 5.2 ± 1.0 %.
Virginia Biodiesel Refinery (West
Point,
VA,
USA)
and
Seattle
Biodiesel (Seattle,WA, USA)
84%
16% of impurities containing soap,
Docosahexaenoic acid (4.91 g l-1)
(Chi et al., 2007)
Animal
(Nitayavardhana et
free fatty acids, excess methanol,
unreacted
triglycerides,
diglycerides, or monoglycerides
Se Quential Pacific Biodiesel, Inc.
__
(Salem, OR, USA)
biomass
feed
(0.83±
0.02
-1
increase-g
g
initial
al.,
2011)
biomass)
Biofuel
Research
Pte.
Ltd.
Glycolipid (3.91 g-l-1 d-1)
__
(Singapore)
83
(Yanbin Liu et al., 2011)
Glycerol
Impurities
present
in
crude
content
glycerol
__
6
Ref.
(w/w)
Rothsay, Guelph, ON
Biocapital, Charqueada, SP, Brazil
Biodiesel factory, Portugal
70–75%
__
86%
mol-H2
-1
mole
glycerol
(Sabourin-Provost et al.,
consumed
2009)
__
0.53 kg-H2 .100 m-3 h-1
(Fernandes et al., 2010)
MONG 6.2% (w / w), ash (4.6% w /
710.0 cm -H2dm medium
v) and methanol (0.03% w /w)
84
3
-3
(Marques et al., 2009)
Table 2.1. 2: Maximum hydrogen production by different inocula using different non-glycerol feedstock (Hak-Joo Kim et al., 2004)
Substrate
Inocula
Operation
Maximum hydrogen yield
Ref.
Mode
-1 -1
Micro-flora of fermented soybean-meal
Batch
130 ml-H2 h l culture
(Mizuno et al., 2000)
Food waste
Anaerobic sludge
Batch
1.8 mol-H2 mol-1 hexose
(Shin et al., 2004)
Food waste
Anaerobic sludge
Batch
101 ml-H2 g COD
(Chen et al., 2006)
Beer lees
Micro-flora of cow dung compost
Batch
68.6 ml- H2 g-1 total volatile
(Fan et al., 2006a)
Bean
curd
manufacturing
waste
-1
solid
Rice winery wastewater
Municipal sewage sludge
Continuous
1.9 mol- H2 mol-1 hexose
(Hanqing Yu et al., 2002)
Rice slurry
Anaerobic sludge
Batch
346 ml- H2 g-1 carbohydrate
(Fang et al., 2006)
Cornstalk wastes
Micro-flora of cow dung compost
Batch
149.69 ml-H2 g-1 total volatile
(Zhang et al., 2007)
solid
Corn starch hydrolisate
Enterobacter aerogenes
Batch
1.8 mmol-H2 mmol-1glucose
(Perego et al., 1998)
Corn starch hydrolisate
E. coli
Batch
0.36 mmol-H2 mmol-1glucose
Molasses
Enterobacter aerogenes E 82005
Continuous
3.5 mol- H2 mol-1 sugar
(Tanisho et al., 1995)
Palm oil mill effluent
Anaerobic sludge
Batch
6.33 l-H2 l-1 substrate
(O-Thong et al., 2008)
85
Substrate
Inocula
Operation
Ref.
(Yokoyama et al., 2007b)
Mode
Cow dung
Micro-flora of cow dung
Batch
743 ml-H2 kg-1 cow dung
Cow waste slurry
Micro-flora of cow waste slurry
Batch
392 ml-H2 l slurry
(Yokoyama et al., 2007a)
Grass silage
Micro-flora from a full-scale landfill
Batch
16 ml-H2 g -1 volatile solid
(Karlsson et al., 2008)
Wheat straw
cow dung compost
Batch
68 ml-H2 g
Sugarcane bagasse
Caldicellulosiruptor saccharolyticus
Batch
Pig slurry
Sludge
from
a
mesophilic
-1
-1
volatile solid
(Fan et al., 2006b)
19.6 ml-H2 g volatile solid
-1
(Ivanova et al., 2009)
Continuous
3.65ml-H2 g -1 volatile solid
(Kotsopoulos et al., 2009)
Continuous
2.9 ml-H2 ml
methanogenic reactor
Cheese whey
Indigenous micro-flora
-1
cheese whey
(Venetsaneas
2009)
86
et
al.,
Table 2.1. 3: Maximum hydrogen production by different inocula using pure and crude glycerol as feedstock
Substrate
Inocula
Pure glycerol
Halanaerobium
saccharolyticum
Mode of experiment
Maximum Hydrogen yield
Batch
0.62 ± 0.02 mol-H2 mol glycerol
Batch
Ref.
-1
(Kivistö et al., 2010)
-1
(subspecies saccharolyticum)
Pure glycerol
Halanaerobium
saccharolyticum
(subspecies senegalensis)
Pure glycerol
Heat-treated anaerobic sludge,
Continuous
0.6 l-H2 l-1 d-1
(Escapa et al., 2009)
Pure glycerol
Domestic wastewater
Batch
3.9 mol-H2 mol-1 glycerol
Pure glycerol
Klebsiella sp. HE1
Batch
(Wu et al., 2011)
Pure glycerol
Anaerobic digested sludge
Batch
(K Seifert et al., 2009)
Pure glycerol
Different heat-pretreated natural micro-
Batch
11.5–38.1 ml-H2 g-1 COD
(Akutsu et al., 2009)
flora.
Pure glycerol
Enterobacter aerogenes HU-101
Continuous
80 mmol-H2 l-1 h-1
(Ito et al., 2005)
Crude glycerol
Thermotoga neapolitana DSM 4359
Batch
2.73±0.14 mol-H2 mol-1 glycerol
(Ngo et al., 2011)
Crude glycerol
Mixed culture
Batch
(Selembo et al., 2009a)
87
Substrate
Inocula
Mode of experiment
Maximum Hydrogen yield
Ref.
Crude glycerol
Continuous
63 mmol-H2 l-1 h-1
(Ito et al., 2005)
Crude glycerol
Anaerobic sludge
Batch
200 ml-H2 g COD
(Fernandes et al., 2010)
Crude glycerol
Batch
710.0 cm3-H2dm-3 medium
(Marques et al., 2009)
Crude glycerol
Enterobacter aerogenes NBRC 12010
Batch
0.77 mol-H2 mol glycerol
Crude glycerol
domestic wastewater
Batch
0.41±0.1 m -H2 m d
Crude glycerol
Rhodopseudomonas palustris
Batch
6 mol-H2 mol-1 glycerol
(Sabourin-Provost et al.,
-1
-1
3
-3
-1
2009)
88
Table 2.1. 4: Different bioreactor systems used for H2 production using glycerol and other feedstock
Substrate
Bioreactor type
Bioreactor operation conditions
Maximum
Hydrogen
Ref.
yield
◦
Temp. ( C)
Pure glycerol
Small sealed glass tube
37
Optimum
Mode
pH
operation
7.4
Batch
of
0.62 ± 0.02 mol-H2 mol-1
glycerol
Pure glycerol
Small sealed glass tube
37
7
Batch
1.61 ± 0.28 mol-H2 mol-1
glycerol
Pure glycerol
Microbial electrolysis cell(MEC) with a
25
7
Continuous
0.6 l-H2 l-1 d-1
30
7
Batch
(Selembo
250 ml anodic chamber and a gasphase cathode
Pure glycerol
Single-chamber membrane less MEC
reactors consisted of a 4-cm long by 3-
et
al.,
2009b)
cm diameter cylindrical chambers
Pure glycerol
2.5 l fermenter containing 1.5 l media
35
6
Batch
0.345
mol-H2
mol-1
(Wu et al., 2011)
glycerol
Pure glycerol
Glass reactors (60 cc) with working
37
6
capacity of 30 cc
Batch
(K
Seifert
2009)
89
et
al.,
Substrate
Bioreactor type
Maximum
Hydrogen
Ref.
yield
Temp. (◦C)
Optimum
Mode
pH
operation
Batch
11.5–38.1 ml-H2 g COD
Continuous
80 mmol-H2 l-1h-1
(Ito et al., 2005)
Batch
Pure glycerol
120 ml vials containing 50 ml media
35
6.5
Pure glycerol
Packed-bed reactor of 60 ml working
37
pH
volume
Pure glycerol
500 ml serum glass bottles filled with
not
of
-1
controlled
30
6.2
-1
250 ml media
Pure glycerol
670 cm3 flasks containing 290 cm3 of
125 ml serum bottles
et
al.,
et
al.,
2009a)
37
6.5
Batch
medium
Pure glycerol
(Selembo
30
__
Batch
608.7
cm3-H2dm-3
(Marques
medium
2009)
(Sabourin-Provost et
al., 2009)
Crude glycerol
120-ml serum bottles containing 40 ml
75
7.5
Batch
media
Crude glycerol
500 ml serum glass bottles filled with
2.73 ± 0.14 mol-H2 mol-1
glycerol
30
6.2
Batch
250ml of media
Crude glycerol
Packed-bed reactor of 60 ml working
volume
(Ngo et al., 2011)
(Selembo
et
2009a)
37
pH
not
controlled
90
Continuous
63 mmol-H2 l-1h-1
(Ito et al., 2005)
al.,
Substrate
Bioreactor type
Maximum
Hydrogen
Ref.
yield
Temp. (◦C)
Crude glycerol
2-l glass flasks, consisting of 1 l of liquid
25.0±0.5
Optimum
Mode
pH
operation
5.5
Batch
of
-1
(Fernandes
200 ml-H2 g COD
volume
Crude glycerol
al.,
2010)
3
3
670 cm flasks containing 290 cm of
37
6.5
Batch
medium
Crude glycerol
et
710.0
3
cm -H2dm
-3
medium
(Marques
et
al.,
2009)
-1
two-compartment
30
6
Batch
Single-chamber membrane less MEC
30
7
Batch
0.41 ± 0.1 m3-H2 m-3 d-1
(Selembo
Bio-electrochemical
reactor
Crude glycerol
reactors consisted of a 4-cm long by 3-
et
al.,
2009b)
cm diameter cylindrical chambers
Crude glycerol
30
__
Batch
al., 2009)
Other substrates
Bean
curd
manufacturing
1.2-litre serum vial with a 1-litre culture
35±1
6
Batch
130 ml-H 2 h-1l-1 culture
(Mizuno et al., 2000)
55 ±1
5.5
Batch
(Shin et al., 2004)
volume
waste
Food waste
715-ml serum bottles
91
Substrate
Bioreactor type
Maximum
Hydrogen
Ref.
yield
Temp. (◦C)
Optimum
Mode
pH
operation
of
-1
Food waste
250-mL serum bottles
36±1
5.5 ± 0.1
Batch
101 ml-H2 g COD
(Chen et al., 2006)
Beer lees
250-ml serum vials
36±1
6-7
Batch
68.6 ml-H2 g-1 total volatile
(Fan et al., 2006a)
solid
Rice
winery
3.0-l up-flow reactor
55
5.5
Continuous
-1
2.14 mol-H2 mol hexose
wastewater
Rice slurry
(Hanqing Yu et al.,
2002)
37
4.5
Batch
346
g-1
ml-H2
(Fang et al., 2006)
carbohydrate
Cornstalk
250 ml serum vials
36±1
7
Batch
wastes
Corn
149.69
ml-H2
g-1 total
(Zhang et al., 2007)
volatile solid
starch
2.0 l bioreactor with a working volume of
hydrolysate
1.5 l
Corn
2.0 l bioreactor with a working volume of
starch
hydrolisate
1.5 l
Molasses
300 ml hand-made fermenter having
40±0.5
5.5
Batch
1.8
mmol-H2
mmol-1
mmol-1
glucose
37 ± 0.5
5.5
Batch
0.36
mmol-H2
glucose
___
6
250 ml culture fluid
92
Continuous
3.5 mol-H2 mol-1 sugar
(Tanisho et al., 1995)
Substrate
Bioreactor type
Maximum
Hydrogen
Ref.
yield
Temp. (◦C)
Palm oil mill
60
Optimum
Mode
pH
operation
5.5
Batch
of
-1
6.33 l-H2 l substrate
et
al.,
2008)
Effluent
Cow dung
625 ml glass bottle tightly sealed with
60
6.6
Batch
-1
743 ml-H2 kg cow dung
butyl rubber cap
Cow
(O-Thong
waste
2-l fermentor
60
7
Batch
392 ml-H2 l-1 slurry
(Yokoyama
al.,
et
al.,
2007a)
Continuous
stirred
tank
reactors
55
7
Batch
16 ml-H2 g -1 volatile solid
constructed from 2 l-glass bottles
Wheat straw
et
2007b)
slurry
Grass silage
(Yokoyama
250 ml serum vials
(Karlsson
et
al.,
2008)
36 ± 1
7
Batch
68.1 ml-H2 g
-1
volatile
(Fan et al., 2006b)
-1
volatile
(Ivanova et al., 2009)
solid
Sugarcane
Anaerobic 50 ml hypo-vials
70
__
Batch
bagasse
Pig slurry
solid
Continuous stirred tank reactor having
70
__
Continuous
3
750 cm working volume
Cheese whey
19.6 ml-H2 g
500
ml
active
volume
mesophilic
35
5.2
CSTR-type digester
Continuous
3.65 cm3-H2 g-1 of volatile
(Kotsopoulos et al.,
solid
2009)
2.9 ml-H2
whey
93
ml-1 cheese
(Venetsaneas et al.,
2009)
Table 2.1. 5: Microorganisms used for bioconversion of pure and crude glycerol to H2 and other products
Strain (s)
Thermotoga neapolitana DSM 4359
Halanaerobium saccharolyticum
0.62 ± 0.02 mol-H2 mol-1 glycerol
(Ngo et al., 2011)
1,3-propanediol
(PD),
butyrate,
ethanol and 0.58 ± 0.03 mol-CO2
mol-1 glycerol
-1
Acetate and 1.11 ± 0.21 mol-CO2
mol-1 glycerol
senegalensis)
Mixed culture
Ref.
-1
( subspecies Saccharolyticum)
Halanaerobium saccharolyticum (subspecies
Other by-product (s)
Heat treated anerobic sludge
0.6 l-H2 l-1 d-1
63 mmol-H2 l-1h-1
Domestic wastewater
0.41 ± 0.1 m3-H2 m-3 d-1
0.59 mol-PD mol-1 glycerol and 39%
(Selembo
CO2 in gas phase
2009a)
et
al.,
0.85 mol-ethanol mol-1 glycerol
(Ito et al., 2005)
(Selembo
et
al.,
et
al.,
et
al.,
2009b)
Klebsiella pneumoniae ATCC 25955
__
1,3-propanediol
(Barbirato
1998)
Citrobacter freundii ATCC 8090
__
1,3-propanediol
94
(Barbirato
Strain (s)
Ref.
1998)
Enterobacter agglomerans CNCM 1210
__
(Barbirato
1,3-propanediol
et
al.,
et
al.,
1998)
Clostridium butyricum CNCM 1211
__
(Barbirato
1,3-propanediol
1998)
Rhodopseudomonas palustris
-1
6 mol-H2 mol glycerol
__
al., 2009)
Mixed micro flora obtained from fixed-bed
200 ml-H2 g-1 COD
(Fernandes
__
anaerobic reactors
et
al.,
2010)
710.0 cm3-H2dm-3 medium
345.0 cm3-CO2dm-3 medium
(Marques
et
al.,
2009)
Enterobacter aerogenes NBRC 12010
0.80 mol-CO2 mol-1 glycerol
Mixed micro flora of organic waste or soil
11.5–38.1 ml-H2 g-1COD
1,3-propanediol
Klebsiella sp. HE1
Anaerobic digested sludge
-1
(Wu et al., 2011)
-1
0.784 ± 0.063 l-CO2 l media
(K
Seifert
2009)
95
et
al.,
Strain (s)
Ref.
Klebsiella pneumoniae
__
53 g-PD l–1 crude glycerol
(Mu et al., 2006)
Pichia pastoris
__
Phytase
(Tang et al., 2009)
Microalga Schizochytrium limacinum
__
Docosahexaenoic acid
(Chi et al., 2007)
Edible fungus, Rhizopus microsporus
__
Animal feed
(Nitayavardhana
et
al., 2011)
Ustilago maydis
__
Glycolipids
(Yanbin Liu et al.,
2011)
96
Raw materials
-Vegetable oils
-Animal fats
Methanol
Heat
Transesterification
Agitation
Sodium Hydroxide
Phase Separation
Distillation
Crude Glycerol
Biodiesel
Figure 2.1.1: Schematic representation of a general biodiesel production process using
transesterification of vegetable oil and fats (adapted from (Hak-Joo Kim et al., 2004, Muniyappa et
al., 1996, Schuchardt et al., 1998))
Figure 2.1.2: Equation showing transesterification of large branched triglyceride molecule to
biodiesel and glycerol (Muniyappa et al., 1996)
98
Europe
Americas
Asia-pacific
Others
Figure 2.1.3: Global biodiesel production share of different geographic regions of the world till
2009
99
Figure 2.1.4: Schematic representation of glycerol metabolism during anaerobic fermentation
(Adapted from (Biebl et al., 1999, Drożdżyńska et al., 2011))
100
Figure 2.1.5: Stoichiometric equations showing hydrogen yield during glycerol bioconversion
101
Figure 2.1.6: A two-chambered microbial electrolysis cell used for H2 production by bioconversion
of glycerol (Escapa et al., 2009, Hongqiang Hu, 2009, Hong Liu et al., 2005, Logan et al., 2008)
102
Figure 2.1.7: Anaerobic metabolism of glucose and pyruvate and hydrogen production (Adapted
from (Chin et al., 2003, Mathews et al., 2009)
103
PART-II
KEY ENZYMES IN VALUE-ADDITION OF CRUDE GLYCEROL TO
BIOHYDROGEN
Saurabh Jyoti Sarma1, Gurpreet Singh Dhillon1,
Satinder Kaur Brar1, Yann Le Bihan2 and Gerardo Buelna2
1
Institut National de la Recherche Scientifique (INRS)
Centre Eau, Terre & Environnement, Québec(QC), Canada
2
(Corresponding author, Phone: 1 418 654 3116; Fax: 1 418 654 2600; E-mail:
[email protected])
Ce chapitre de livre a dû être retiré de la version électronique en raison de
restrictions liées au droit d’auteur.
Enzymes in value-addition of waste (2014)
105
PART-III
HYDROGEN BIOREFINERY: POTENTIAL UTILIZATION OF THE LIQUID
WASTE FROM FERMENTATIVE HYDROGEN PRODUCTION
Saurabh Jyoti Sarmaa, Vinayak Pachapura, Satinder Kaur Brara, Yann Le Bihanb,
Gerardo Buelnab
a
Institut National de la Recherche Scientifique (INRS), Centre Eau, Terre &
Environnement, 490, Rue de la Couronne, Québec(QC), G1K 9A9, Canada
b
Centre de Recherche Industrielle du Québec (CRIQ), Québec(QC), G1P 4C7, Canada
[email protected])
Renewable and Sustainable Energy Reviews (Submitted)
141
Résumé
En termes de potentiel de réduction des émissions de gaz à effet de serre, l'hydrogène
est supérieur aux biocarburants commerciaux et aux combustibles fossiles, car il a une
densité d'énergie élevée et il génère principalement que de l'eau. La production de
biohydrogène a un avantage environnemental supplémentaire puisque les différents
déchets organiques peuvent être valorisés au cours du processus. Cependant, en
raison du coût élevé du procédé de bioconversion, sa production commerciale n'est pas
encore réaliste.
Lors de la fermentation sombre, en plus de l'hydrogène, environ 60% de la charge
d'alimentation peut être convertie en divers produits chimiques industriels, y compris
l'éthanol, le 1-3 propanediol et l'acide butyrique. Si ces substances ne sont pas
récupérées, les déchets liquides générés pendant le processus peuvent être utilisés
comme matière première pour la production de polyhydroxyalcanoates, des lipides, du
méthane, de l'hydrogène et de l'électricité. Le rejet liquide est aussi un substitut
potentiel des engrais biologiques de solubilisation du phosphate. Ainsi, dans cette
étude, le processus de production de biohydrogène est évalué comme une bioraffinerie
potentiel pour la production de biocarburants, la chimie fine et des biomatériaux. La
stratégie pourrait être utile pour réduire le coût global du procédés en générant des
revenus de sources multiples.
Mots clés: biohydrogène; bioraffinerie; acide butyrique; éthanol; 1, 3 propanediol;
déchets liquides
142
Abstract
In terms of greenhouse gas emission reduction potential, hydrogen is superior to
commercial biofuels and fossil fuels because, it has high energy density and it
generates only water as major emission. Biohydrogen production has additional
environmental benefit as different organic wastes can be valorized during the process;
however, because of high process cost, its commercial production is yet to start. During
dark fermentation, in addition to hydrogen, around 60 % of the feedstock may convert to
various industrial chemicals including ethanol, 1, 3 propanediol and butyric acid. If these
products are not recovered, the liquid waste generated during the process can be used
as the feedstock for production of polyhydroxyalkanoates, lipid, methane, hydrogen and
electricity. The liquid waste is also a potential substitute of phosphate solubilizing
biofertilizers. Thus, in this review, biohydrogen production process is evaluated as a
potential biorefinery producing biofuels, fine chemicals and biomaterials. The strategy
could be useful to reduce overall cost of the process by generating revenue from
multiple sources.
Key words: Biohydrogen; biorefinery; butyric acid; ethanol; 1, 3 propanediol; liquid
waste
143
Introduction
Depleting fossil fuel reserves and the concern of global climate change have
accelerated the development of renewable energy sources. As a result of greenhouse
gas emission reduction awareness and significant success in the field of renewable
energy research, presently, some of the renewable energy carriers are being used in
commercial level. Bioethanol, biodiesel and biogas are the common non-fossil
renewable fuels derived from biomass. Apart from biofuels, biomass based feedstock
can also be used for production of chemicals or biomaterials. In future, mostly due to
unavailability of raw materials, petroleum based refinery, presently involved in the
production of fuels, chemicals and materials; will have to reduce their production level.
In this context, biorefinery has emerged as a potential alternative of petroleum based
refinery. Biorefinery is a concept where biomass is used as an alternative of petroleum
based raw materials and a range of products such as biofuel, industrial chemicals and
biomaterials including commercially important biopolymers are produced.
Biohydrogen is a biofuel which has different advantages over presently commercialized
biofuels such as, bioethanol, biodiesel and biogas. It is a cleaner energy carrier
releasing water and negligible amount of nitrogen oxides during its combustion. In terms
of greenhouse gas emission reduction, therefore, it has tremendous advantage over
fossil fuels as well as other biofuels. Compared to petrol, a vehicle using CNG
containing 30 % of hydrogen can reduce the particulate matter emission by 55 %,
nitrogen oxides and hydrocarbon (non-methane) emission by 60 %, and CO2 emission
by
35
%
http://ec.europa.eu/dgs/jrc/index.cfm?id=1410&obj_id=17140&dt_code=NWS&lang=en
&ori=HLN (accessed on 31/01/2014). Although, relatively low volumetric energy density
is one of major technological concerns of hydrogen fuel; the energy yield of hydrogen is
122 KJ/g, which is nearly 2.75 times higher than that of conventional hydrocarbon
based fuels (Chang et al., 2004). Biohydrogen can be produced from any biomass,
ranging from those used as food/feed, forest biomass, algae/fungi to agro-industrial
wastes. Dark fermentation is commonly used, rapid and relatively simple method of
biohydrogen production. However, during hydrogen production by this method, only 30-
144
40 % of the substrate is utilized in hydrogen production and remaining 60-70 % are
converted to various other metabolites (Venkateswar Reddy et al., 2013). Ethanol,
butyric acid, and 1, 3 propanediol are some commercially attractive metabolites
produced during hydrogen production. Acetic acid, succinic acid, lactic acid and
propionic acid are a few other metabolites accumulated during hydrogen fermentation. If
these products are not recovered, the fermentative hydrogen production liquid waste
(HPLW)
can
be
converted
to
other
value-added
products
such
as,
polyhydroxyalkanoates (bioplastic) (Venkata Mohan et al., 2010), lipid (Venkata Mohan
et al., 2012), biobutanol (WH Chen et al., 2011), methane and even hydrogen (Su et al.,
2009b). Additionally, organic acid containing HPLW is a potential substitute of
phosphate solubilizing bio-fertilizer (Sarma et al., 2013b). Therefore, the concept of
hydrogen production from biomass based substrate can be extended to hydrogen
biorefinery; where, in addition to hydrogen, different other products of commercial
interest could be simultaneously produced (Figure 2.3.1). Incomplete substrate
utilization is one of the major drawbacks of dark fermentative hydrogen production. The
proposed strategy will help to get rid of this disadvantage by ensuring maximum
biomass utilization. On the same note, it will generate additional revenue to support
biological hydrogen production, industrial production of which is otherwise not possible
due to high process cost.
Characteristics of the liquid waste from biohydrogen production
process
HPLW from crude glycerol (CG) based hydrogen production process has been
characterized for organic carbon content, total nitrogen, dry cell weight, elemental
composition, and pH (Sarma et al., 2013c). It has been observed that biohydrogen
production using 1 Kg of CG can generate as high as 400 L of HPLW (Sarma et al.,
2013a). According to this estimation, production of 1 Kg of hydrogen may generate as
high as 8771.92 L of HPLW (Sarma et al., 2013a). Depending on the substrate used,
COD of HPLW may be as high as 55±1.5 % of original COD (75,200–96,300 mg/L) of
the substrate (O-Thong et al., 2008). As a result of acidic byproduct accumulation,
HPLW generally has a low pH which may be as low as 3.1 (Sung et al., 2002). This pH
145
value may vary according to initial pH of the medium, process mode and duration, and
microorganism selected for the process. In this context, how the acidic pH affects valueaddition or disposal of HPLW will be interesting to know. It must be explored whether
such a low pH will be helpful for cost competitive recovery of certain byproducts. Based
on the type of substrate, microorganism used and process mode applied; the
composition of HPLW may also vary. Major variation may be observed in terms of the
types of the fermentation end products, their concentrations as well as the residual
substrate concentration. As mentioned in the introduction section, acetic acid, butyric
acid, propionic acid, succinic acid, lactic acid, ethanol and PD are some of the
metabolites which may be present in the HPLW (Rajhi et al., 2013, Selembo et al.,
2009). Reported concentrations of different metabolites in HPLW and their market price
are presented in Figure 2.3.2 (Han et al., 2010, Lalaurette et al., 2009, Selembo et al.,
2009). It has been observed that all the metabolites have very high commercial value
and the amount of PD, ethanol, acetic acid, butyric acid, and succinic acid present in
HPLW is worthy to be recovered. In this context, it should be noted that the market
prices of different byproducts of hydrogen production process mentioned in Figure 2.3.2
are the prices of their highly pure form. It implies that these are the maximum
commercial values of such products one can expect; however, very high purification
cost may be the major bottleneck in recovering such pure products. Therefore, proper
cost benefit analysis of the recovery process of each of these products is a prerequisite
of proposed hydrogen biorefinery approach. In Figure 2.3.3, a comparison of Gibbs free
energies of formation (∆Gfº) for different byproducts of hydrogen production process is
presented
https://www.e-
education.psu.edu/drupal6/files/be497b/pdf/Bioenergetics_AppA.pdf
(01/02/2014)http://highered.mcgrawhill.com/sites/dl/free/0072849606/315014/physical_properties_table.pdf (accessed on
01/02/2014). Figure 2.3.3 showed that among different byproducts, succinic acid has
highest tendency to be produced. For microbial processes, however, the enzymatic
pathways exist in the microorganism and the levels of expression of different enzymes
are the crucial factors to determine the type and amount of different products. Hence,
succinic acid may not be the dominant byproduct of a process chosen/optimized for
146
maximum hydrogen production. Moreover, hydrogen may be utilized during succinic
acid production (Krishnakumar, 2013, Murarka et al., 2008); which, in turn may reduce
the hydrogen yield. Therefore, in order to develop a hydrogen based biorefinery, the
process should be designed in such a way that the hydrogen production is not
compromised due to the production of particular byproduct. In next sections, potential
utilization of HPLW for the recovery of certain important products or its potential
utilization as a feedstock for the production of different valuable products has been
elaborately discussed.
Ethanol recovery from HPLW
Ethanol produced from renewable materials is a commercially successful biofuel. Since
1980s, bioethanol produced from corn has been used by blending with gasoline (Sun et
al., 2002). For conventional internal combustion engines, gasoline with 5-26%
anhydrous ethanol can be used as fuels (Gnansounou et al., 2005); however, suitable
modification of the engine is required to use only hydrated ethanol (approximately 95%
ethanol and 5% H2O) as the fuel. The average octane number or the anti-knock index
(AKI) of ethanol is 99; which is significantly higher than that of (88) gasoline
(Gnansounou et al., 2005). In order to increase the octane number of gasoline,
conventionally, methyl tertiary butyl ether (MTBE) is added; however, it is known to be
toxic and a potential contaminant of groundwater (Sun et al., 2002). Alternatively,
ethanol could be used as an eco-friendly replacement of MTBE.
For bioethanol production, the common feedstock, such as sugarcane, corn and wheat
and barley are used by the commercial biofuel producers in Brazil, US or Europe (Balat
et al., 2009). Similarly, cassava, sweet sorghum, sweet potatoes, and lignocellulosic
biomass are the other common substrates (Balat et al., 2009). Alternatively, as
mentioned in the introduction section, ethanol can also be recovered from the liquid
waste of dark fermentative hydrogen production. Table 2.3.1 summarizes a number of
studies on simultaneous hydrogen and ethanol production. From Table 2.3.1, it can be
concluded that the amount of ethanol and hydrogen produced during dark fermentative
hydrogen production varies from 0.29 mol ethanol and 1.04 mol H2/mol glucose to 0.90
mol ethanol (equivalent to 0.23g ethanol/g glucose) and 1.58 mol H2/mol glucose (Ken147
Jer Wu et al., 2007, Zhao, 2009). Even hydrogen production as high as 2.49 mol H2/mol
glucose is also known where fermentation end-product was mostly ethanol (You-Kwan
Oh et al., 2003). Moreover, hydrogen and ethanol yield may vary according to the type
of substrate as well as microbial strain and process mode used for the purpose. For
example, production of 0.96 mol ethanol and 1.12 mol H2/mol-glycerol has been
reported for Enterobacter aerogenes HU-101(Ito et al., 2005). From Table 2.3.1, it is
also clear that the microbial agents used for the purpose are either facultative/strict
anaerobic bacteria or anaerobic mixed culture, which could be thermophilic or
mesophilic. Glucose, α-cellulose, acid treated cellulose hydrolysates, cellobiose, xylose,
sucrose, fructose, pure glycerol, biodiesel manufacturing process liquid waste
containing glycerol, molasses or wastewater containing molasses are the different
substrates evaluated for simultaneous hydrogen and ethanol production. For
comparison purpose, different dedicated bioethanol production processes with no
hydrogen production have been summarized in Table 2.3.2. From the table it is clear
that unlike, simultaneous hydrogen and ethanol production mostly yeasts are used for
dedicated bioethanol production processes. Similarly, the ethanol yield of different
processes listed in Table 2.3.2 ranges from 0.41 g/g glucose to 0.48 g/g substrate.
These values are nearly twice the yield of 0.90 mol ethanol/mol glucose (equivalent to
0.23g ethanol/g glucose) known for simultaneous hydrogen and ethanol production.
Thus, the ethanol yield of a simultaneous hydrogen and ethanol production process is
obviously lesser than a dedicated process. In the former case, apart from ethanol,
hydrogen is also produced and the fermented broth containing ethanol and other
byproducts is mainly considered as waste. Therefore, although the ethanol yield of a
hydrogen production process is not comparable to that of a dedicated ethanol
production process, still recovery of such ethanol may be helpful in reducing the overall
process cost of hydrogen production. However, this possibility needs to be verified by a
thorough cost benefit analysis of simultaneous hydrogen and ethanol production and
purification. At present number of reports are available only on concomitant hydrogen
and ethanol production; whereas, ethanol purification from HPLW is largely unexplored.
148
1,3-propanediol recovery from HPLW
1, 3-properndiol (PD) is an important platform chemical with global production of more
than 200,000 tons per annum (Szymanowska-Powałowska et al., 2013). Different
commercially important applications of PD are as follows: (i) polytrimethylene
terephthalate, a biodegradable polymer widely used in textile fiber production can be
produced from PD (Dabrowski et al., 2012, Drożdżyńska et al., 2011, Saxena et al.,
2009), (ii) it is also used as monomer for the production of different other polymers,
such as polyurethane, and polyether (Barbirato et al., 1995, Drożdżyńska et al., 2011,
Ferreira et al., 2012), (iii) it is an important material for the production of different
cosmetics, detergents, and adhesives (Drożdżyńska et al., 2011, Ferreira et al., 2012)
and (iv) as solvent and for production of dioxanes (Biebl et al., 1999).
PD can be produced by chemical synthesis and biotechnological methods. These
methods comprise: (i) catalytic synthesis using ethylene oxide, carbon monoxide, and
hydrogen (Dabrowski et al., 2012, Saxena et al., 2009), (ii) chemical synthesis using
acrolein (Drożdżyńska et al., 2011, Ken-Jer Wu et al., 2007), (iii) bioconversion of crude
glycerol (Drożdżyńska et al., 2011, Ferreira et al., 2012, Selembo et al., 2009), and (iv)
bioconversion of glucose (Biebl et al., 1999). Out of these methods, utilization of
renewable materials, no toxic byproduct formation, no need of any catalyst,
simultaneous treatment of industrial waste, and mild process conditions are some of the
benefits of microbial PD production. Additionally, as shown in Table 2.3.3, during PD
production by bioconversion of organic materials, such as pure/crude glycerol, hydrogen
can be recovered as a byproduct or vice-versa. Therefore, liquid waste generated
during fermentative hydrogen production using pure/crude glycerol could be considered
as a potential renewable source of PD. Crude glycerol is a byproduct of biodiesel
manufacturing by trans-esterification process and nearly 10 Kg of crude glycerol is
generated for every 100 Kg of biodiesel produced (Santibáñez et al., 2011). Although it
is an expensive process (Santibáñez et al., 2011); glycerol can be recovered from crude
glycerol (Yong et al., 2001). Owing to the increase in global biodiesel demand, crude
glycerol production is increasing day by day. In turn, due to availability of large amount
of crude glycerol, compared to demand, production of glycerol is increasing. Therefore,
149
a sharp decrease in glycerol price has been seen (Chunfei Wu et al., 2013). Thus,
considering all these facts, hydrogen production by crude glycerol bioconversion and
subsequent recovery of PD form the liquid waste stream could be an interesting option.
As shown in Table 2.3.3, using crude glycerol, simultaneous production of 0.31 molH2/mol-glycerol and 0.59 mol-PD/mol-glycerol is possible. Therefore, from 1 Kg crude
glycerol (derived from trans-esterification of soybean oil) containing 63 % (w/v) of
glycerol (Hu et al., 2012), nearly 48.14 L (2.12 mol) of H2 and 307.06 g (4.03 mol) of PD
could be produced. Additionally, aforementioned crude glycerol also contains nearly 37
% (w/v) of non-glycerol materials; therefore, actual hydrogen and PD production from 1
Kg of crude glycerol could be more that the projected value. Present market price of
analytical
grade
PD
is
https://www.fishersci.ca/coupon.do?cid=4509876&lang=en&itemId=
$288/L
(accessed
on
06/02/2014). Likewise, based on cylinder size, the current (2013) market value of
industrial
grade
hydrogen
is
around
$7.75-$11.50/cubic
meter
http://infopage.gem.wa.gov.au/docs/Price_Schedule_-_Industrial_Gases_49009.xls?
(accessed on 17/02/2014). Therefore, commercial potential of simultaneous recovery of
both the products by an eco-friendly process such as bioconversion of organic waste
needs to be explored.
Butyric acid recovery from HPLW
Butyric acid has different commercially important applications which include: (i)
production of cellulose acetate butyrate fiber suitable for textile industry (Zhang et al.,
2009), (ii) potential application as a raw material for biotechnological production of
butanol (Dwidar et al., 2012), (iii) preparation of polymeric fiber with enhanced heat,
sunlight and cold resistance (Dwidar et al., 2012, Zhang et al., 2009), (iv) for bio-plastic
(β-hydroxybutyrate) production (Dwidar et al., 2012, Zhang et al., 2009), (v) for
improved biobutanol production efficiency (Tashiro et al., 2004, Xuepeng Yang et al.,
2013, Zhang et al., 2009), (vi) for the production of fuels such as ethyl butyrate or butyl
butyrate (Dwidar et al., 2012, Salihu et al., 2013), (vii) as calcium butyrate for leather
tanning purpose (Zhang et al., 2009), (viii) as tributyrin for potential treatment of
malignancy (Zi-Xing Chen et al., 1994), (ix) butyric acid esters as aroma compounds
150
and flavoring agent (Dwidar et al., 2012, Larios et al., 2004, Zhang et al., 2009), and (x)
as indole-3-butyric acid- a plant hormone. Butyric acid is commonly produced by
butyraldehyde oxidation (Zigova et al., 2000). The starting material for this process is
propylene, which in turn is synthesized from crude oil (Dwidar et al., 2012). However,
this method has certain disadvantages: (i) as it is a chemical process, the butyric acid
produced by this method is not preferred for the processes where the product of natural
origin is required, (ii) propylene is one of the mostly used crude oil derived raw materials
having wide range of other applications; therefore, by using renewable sources for
butyric acid production considerable amount of propylene could be saved and rout to
other more valuable products, and (iii) crude oil is not a renewable resource; therefore,
there is a need to look into other more sustainable opportunities. In this context, organic
acid rich liquid waste of dark fermentative hydrogen production could be considered as
a renewable and less expensive source of butyric acid. As shown in Table 2.3.4, during
hydrogen production by bioconversion of organic materials/organic waste materials, the
amount of butyric acid accumulated in the fermentation medium could be as high as
61.0 % of total soluble microbial products (Abreu et al., 2009). Apart from producing 3.2
mol-H2/mol-hexose, by weight the amount of butyrate produced as the byproduct of the
process could be as high as 3793 ± 671 mg-butyrate/L (Hafez et al., 2009). Similarly,
hydrogen and butyrate yield as high as 2.59 mol-H2/mol-hexose and 0.62 molbutyrate/mol-hexose have been recorded during bioconversion of the wastewater from
sugar factory (Ueno et al., 1996). From these observations it could be concluded that by
bioconversion of 1 Kg of glucose (5.55 mol); at the same time and at same production
cost, nearly 28.75 g (326.44 L at STP) of hydrogen gas and 299.37 g of butyrate can be
produced. As already mentioned, present market value of hydrogen is around $7.75$11.50/cubic meter. Similarly, present market value of bulk quantity of natural butyric
acid
is
around
$2-$50/Kg
gs/703515617/natural_Butyric_acid_107_92_6.html
http://www.alibaba.com/product(accessed
on
30/10/2013).
Whereas, the price of analytical grade butyric acid could be as high as $74.6/L
https://www.fishersci.ca/coupon.do?cid=AC108110010&lang=en&itemId=,AC10811001
0 (accessed on 06/02/2014). Therefore, instead of concentrating on production of only
hydrogen or only butyric acid, in order to ensure maximum utilization of the feedstock as
151
well as to simultaneously reduce the production cost of both the products; more
investigations should be carried out for developing an efficient integrated hydrogen and
butyric acid production process. From Table 2.3.4 and above discussion it is clear that a
significant amount of hydrogen and butyric acid could be produced by an integrated
bioconversion process by using either pure feedstock such as glucose or even highly
complex materials such as industrial wastewaters. Therefore, instead of merely trying to
improve the yield of one product; more effort should be given to develop an efficient
process for simultaneous industrial production of both the products. Similarly, instead of
concentrating on reducing only production cost or only purification cost of hydrogen and
butyric acid; more effort should be given to reduce total production and purification cost.
For example, as shown in Table 2.3.4, to reduce the process cost instead of expensive
feedstock, such as glucose or sucrose industrial waste materials, such as corn syrup
waste and wastewater from sugar factory have been used. However, application of a
relatively complex feedstock, such as wastewater may increase the purification cost of
butyric acid by complicating the purification process. Similarly, in order to bypass the
sterilization of the feedstock required for a process involving pure microbial culture,
processes with mixed microbial culture are often considered. However, in the case of
mixed culture based processes, number of different gaseous and liquid products will be
produced. Thus, there is a chance that product purification cost will increase.
Methane production from HPLW
Compared to methane, hydrogen is more valuable fuel. Therefore, although methane
production from organic waste material is a traditional practice, yet hydrogen production
is another commercially significant option. Instead of combusting methane alone,
application of a mixture of H2 and methane can reduce nitrogen oxide emission (Zhu et
al., 2008). As already discussed, unlike methane production, during fermentative
hydrogen production different solvents and organic acids such as ethanol, butanol, PD,
acetic acid, and butyric acid are produced as fermentation end products. These end
products can be further converted to methane by conventional anaerobic digestion.
Alternatively, both hydrogen and methane production can be simultaneously carried out
in a single process; however, optimum hydrogen and methane production conditions
152
are not exactly same (Zhu et al., 2008). Likewise, in a single-stage hydrogen and
methane production process, methane producers (methanogens) can consume
hydrogen. Therefore, methane recovery from hydrogen production liquid waste by a
two-stage hydrogen and methane production process has obvious technical advantage
over only methane production or single-stage methane and hydrogen production using
organic waste. In Table 2.3.5 some recent studies on two-stage hydrogen and methane
production have been summarized. From the table, it is evident that continuous
hydrogen and methane production process is quite popular among the researchers.
One major reason for such preference is that a continuous process can prevent the
accumulation of organic acids produced as byproducts of hydrogen production; thereby,
early termination of the process due to sharp pH drop can be avoided. Additionally,
decrease in medium pH may initiate metabolic shift to reduce the cumulative hydrogen
production (Khanal et al., 2004). Likewise, mixed microbial culture from waste material
is the preferred inoculum over pure strains. Additionally, experiments have been
commonly carried out at larger scale using 10L to 500L reactors. It indicates that
considerable success that has been made in the field of biomethane production using
HPLW. However, major drawback of this strategy is that valuable acids and solvents
present in HPLW are converted to methane, which otherwise, would have been
recovered.
Bioplastic (PHA) production from HPLW
Polyhydroxyalkanoates (PHAs) are the polyesters which can be produced by
microorganisms by utilizing organic substrates including waste materials (Balaji et al.,
2013, Du et al., 2012, Zinn et al., 2001). Considering the depletion of fossil based
feedstock, PHAs produced from biomass or agro-industrial waste based substrates
have significance in terms of sustainability. Additionally, owing to their biodegradability,
in comparison to petroleum based polyesters they are more eco-friendly. In addition to
biodegradability, biocompatibility is the other important feature of PHAs which make
them suitable for biomedical applications such as tissue engineering (Guo-Qiang Chen
et al., 2005, Samy et al., 2013). PHAs are presently commercialized as low volume high
value products and they have the potential to be used for packaging and textile industry
153
(Keshavarz et al., 2010). Presently, pure substrates, such as glucose, soybean oil (He
et al., 1998); biomass based feedstock, such as hemicellulose (Ramsay et al., 1995),
forest biomass (Keenan et al., 2006); industrial waste materials such as paper industry
wastewater (Bengtsson et al., 2008), molasses (Albuquerque et al., 2007) are known to
be used for microbial polyester, such as PHAs production.
In this context, the liquid waste generated during biohydrogen production process could
be a potential substrate for PHAs production (Patel et al., 2011, Venkata Mohan et al.,
2010). As already an elaborate description has been made, during fermentative
hydrogen production, volatile fatty acids (VFAs), such as acetic acid and butyric acid are
produced as byproducts. Different microorganisms can convert these VFAs to
biopolymers, such as PHAs (Patel et al., 2012, Venkateswar Reddy et al., 2013).
Application of VFAs containing liquid of biohydrogen production process has certain
advantages. Firstly, the waste contains large amount of acetic and butyric acid and their
conversion to structurally close PHAs will be relatively convenient, as simpler metabolic
pathways with fewer number of steps will be involved (Venkata Mohan et al., 2010).
Secondly, COD of the liquid waste will be significantly removed and its acidic pH will be
neutralized by conversion of the organic acids, which in turn will omit treatment
requirement for hydrogen production liquid waste. Thirdly, biomass based feedstock
needs proper pre-treatment for PHAs production (Bowers et al., 2013); however, if the
liquid waste is used, other than the pH adjustment, no other pre-treatment will be
needed. Considering the fact that high process cost is the major bottleneck of
fermentative hydrogen production; the proposed approach has the potential to provide
an additional source of revenue for hydrogen manufacturers to improve the economic
feasibility of the process. Additionally, the technology can offer ecofriendly/sustainable
and renewable polyester in the form of PHAs.
As per the authors’ knowledge, presently only four reports are available on this topic
(Patel et al., 2011, Porwal et al., 2008, Venkata Mohan et al., 2010, Venkateswar Reddy
et al., 2013). Therefore, further investigations are encouraged which should determine
maximum theoretical PHAs production potential of the hydrogen fermenter liquid waste
(HPLW), technological barriers in achieving that limit as well as the process cost
involved. According to Reddy et al. (2013), during fermentative hydrogen production by
154
using organic feedstock, 60-70 % of the organic materials are left as fermentation endproducts (Venkateswar Reddy et al., 2013), most of which are organic acids. Thus, it
will be interesting to know how much of this organic acid could be converted to PHAs
and what is the rate of conversion.
Organic acids present in HPLW may be inhibitory for the microorganism used for PAHs
production (Venkateswar Reddy et al., 2013). In this context, application of acid tolerant
PHAs producing recombinant strain could be considered as a potential strategy for
industrial production of PHAs using HPLW. Due to similar reason, HPLW will not be
suitable for PHAs production by using batch process. Therefore, more attention should
be given to continuous or fed-batch fermentation of HPLW. Chemical analysis of PHAs
produced from HPLW was found to have around 89 % of hydroxy butyrate and 9 %
hydroxy valerate (Venkateswar Reddy et al., 2013). The composition of HPLW having
relatively higher amounts of acetic and butyric acid and relatively lower amount of
propionic acid have been mentioned as a probable reason for high hydroxy butyrate
content (Venkateswar Reddy et al., 2013). The copolymer made up of hydroxy butyrate
and hydroxy valerate has better physical and thermal properties than the homo polymer,
poly-hydroxy butyrate (PHB) (Venkateswar Reddy et al., 2013). Therefore, being a
mixture of different organic acids, HPLW has additional advantage as feedstock for
commercially important PHAs production. Porwal et al. (2008) have reported that
microorganisms, such as Bacillus thuringiensis EGU45 can produce as high as 0.63 mol
H2/mol glucose and 435 mg PHB/L of medium (Porwal et al., 2008). Patel et al. (2011)
have reported that under batch cultivation, Bacillus thuringiensis EGU45 can produce
1.67 mol H2/mol glucose and PHB as high as 11.3 % of dry biomass could be produced
by utilizing the waste generated during the process (Patel et al., 2011). Thus, it will be
interesting to investigate continuous H2 production followed by continuous PHAs
production using HPLW.
Potential application of HPLW as phosphate solubilizer
Plants can take up the soluble phosphorus present in soil; however, the amount of
soluble phosphorus present in soil is relatively low (Park et al., 2009). In the form of
different chemical fertilizers, soluble phosphorus can be applied into the agricultural
155
field. However, a significant portion of applied soluble phosphorus reacts with different
cations present in soil and is converted to insoluble forms, such as, Ca3 (PO4)2, FePO4,
and AlPO4 to become unavailable for plants (Park et al., 2009). Moreover, the soil itself
contains insoluble inorganic phosphorus as the salt of calcium, iron and aluminum
(Yadav et al., 2012). Application of phosphate solubilizing bio-fertilizers (PSBs) is one
method commonly used to increase phosphorus availability of soil. Commercially used
PSBs are the microorganisms which can solubilize the insoluble phosphates present in
soil to make them available for the utilization of plants. These microbes can release
different organic acids which, in turn, chelate the cations of insoluble inorganic
phosphorus present in soil to release bioavailable phosphorus (YP Chen et al., 2006).
As described in previous sections, during hydrogen production by dark fermentation a
range of organic acids are produced; hence, HPLW containing different organic acids
could be used as an alternative of PSB. Laboratory study using insoluble Ca3 (PO4)2
have shown that application of HPLW can increase the soluble phosphorus
concentration by nearly 36 times (Sarma et al., 2013b). It indicates that the approach
has tremendous potential as a substitute of commercial PSBs. Conventional PSBs have
relatively shorter shelf-life of nearly 6 months, because they are nothing but some
microbial cells; viability of which might be lost during storage. On the contrary, HPLW
could have superior shelf-life as it has no relation with microbial cell viability (Sarma et
al., 2013b). Similarly, HPLW is a waste material which has no direct production cost;
however, PSB production process needs expensive chemicals. In general, PSB
formulations are comprised of microbial cell, nutrient and carrier material; whereas,
HPLW could be applied in agricultural field without further formulation. Thus, HPLW has
certain advantages over conventional PSBs; however, its practical applicability should
be verified by field trials. Residual COD and BOD of HPLW might be a concern over its
potential application to the agricultural field. Therefore, prior to its land application,
removal of microbial biomass might be needed.
Other products from HPLW
A report is available on lipid production by microalgae using HPLW as the substrate.
According to Mohan et al. (2012), from each mL of volatile fatty acids (VFAs) rich
156
HPLW, as high as 1.42 mg of wet algal biomass could be produced of which 26.4% are
lipids of commercial interest (Venkata Mohan et al., 2012). This study demonstrated
possible application of HPLW for lipid production. However, algae are capable of
producing as high as 3.2 g biomass /L medium, which is higher than the amount
reported for HPLW (Li et al., 2008). Therefore, further investigation should be
undertaken to achieve maximum biomass/lipid production using HPLW. As already
mentioned, HPLW contains only 60-70 % of the organic matter used for hydrogen
production. Hence, better results can be expected by using HPLW as a co-substrate
with another substrate commonly used for algal lipid production. HPLW can also be
used as a feedstock for hydrogen production by photo-fermentation (Chun-Yen Chen et
al., 2008, Su et al., 2009a, Honghui Yang et al., 2010). Chen et al. (2008) have reported
that by dark fermentation, hydrogen yield as high as 3.80 mol H2/mol sucrose could be
obtained. If the HPLW generated during the process is used as a substrate for photofermentation, cumulative hydrogen yield could be increased up to 10.02 mol H2/mol
sucrose (Chun-Yen Chen et al., 2008). Thus, in terms of substrate utilization and
hydrogen yield, the approach has very high potential; however, it has various concerns
too. Firstly, for this process a light source is required and the culture vessel should be
designed in such a way that sufficient light is available for the microorganisms. This will
increase the process cost and the process will be difficult to scale up; especially, when
the final product is gaseous hydrogen and requires a close reactor with large
headspace. Secondly, the approach is suitable only for pure substrate, such as sucrose
where the fermentation medium is relatively transparent and light can easily penetrate
through
it.
However,
in
the
case
of
waste
based
feedstock,
such
as
wastewater/wastewater sludge, the fermentation medium is relatively opaque and
hence, due to limited light access, the approach may not be able to show similar
efficiency. Owing to high availability and less expensive nature, agro-industrial waste
and biomass based substrates are recognized as most suitable for potential industrial
production of hydrogen. Thus, the approach will have limited industrial significance.
Thirdly, before starting the photo-fermentation, sterilization of HPLW may be needed;
which will increase the process cost and energy consumption. Additionally, compared to
dark fermentation, photo-fermentative hydrogen production process is relatively slow
157
and may take as long as 7-8 days to complete a batch process (Sabourin-Provost et al.,
2009). Hence, this strategy needs a thorough techno-economic analysis to determine its
commercial feasibility. Akin to photo-fermentation, HPLW can also be used as a
substrate to produce hydrogen by using microbial electrolysis cell (MEC) (Lalaurette et
al., 2009). However, requirement of expensive catalyst, such as platinum is one
disadvantage of this approach. Performance of a microbial electrolysis cell may be
reduced by the pH gradient established between anode and cathode chamber
(Rozendal et al., 2008). On a similar note, efficiency of the hydrogen producing
microorganism present in the anode may be altered by the presence of hydrogen
consumer such as, methanogen (Hong Liu et al., 2010, Rozendal et al., 2008).
Relatively low proton concentration of a MEC may increase the internal resistance
(Hong Liu et al., 2010). In order to reduce the resistance, the distance between anode
and cathode should be reduced (Hong Liu et al., 2010). Therefore, pilot scale operation
of the process is challenging. Alternatively, HPLW can be directly converted to
electricity by using a microbial fuel cell (SangEun Oh et al., 2005). However, the
approach also has concerns similar to MEC. Electrode should have very large surface
area to support the biofilm of anodophilic microorganisms (Logan et al., 2006). Cathodic
reaction with poor reducing ability is also considered as a limiting factor of electricity
generation by microbial fuel cell technology (Byung Hong Kim et al., 2007). Thus,
application of HPLW for lipid, hydrogen and electricity production are at natal stage and
these technologies will take some time to attain commercial interest.
Concluding statements
In terms of gravimetric energy density and greenhouse gas reduction potential,
hydrogen produced from biomass based feedstock has clear advantage over
contemporary biofuels, such as biodiesel, bioethanol and biomethane.
Dark
fermentation is mostly investigated, straight forward and a rapid method of biohydrogen
production. During hydrogen production by dark fermentation, 60-70 % of the substrate
is channeled to different byproducts. Ethanol, 1,3 propanediol and butyric acid are such
byproducts which have immense commercial importance. If simultaneous hydrogen
production and ethanol recovery are considered; in addition to 1.58 mol H2, 0.90 mol
158
ethanol could be recovered for each mol of glucose. This ethanol yield is equivalent to
0.23g ethanol/g glucose which is almost half of 0.41 g ethanol/g glucose produced by
conventional bioethanol production process. Considering the fact that hydrogen is the
main product and ethanol recovered from HPLW has no direct production cost; the yield
is significant. According to an estimation bioconversion of 1 Kg of crude glycerol, a
waste generated during biodiesel production, can produce 48.14 L of H2 and 307.06 g
of PD. Considering present market value of H2 ($7.75-$11.50/m3) and PD ($2.43/Kg$288/L), the amount of PD which could be recovered from HPLW has industrial
importance. By bioconversion of 1 Kg of glucose, apart from producing 326.44 L of
hydrogen, 299.37 g of butyrate with a bulk price as high as $50/Kg - $74.6/L, can be
recovered as a by-product. During hydrogen production, at the same time more than
one by-product can be recovered from the HPLW. In this case, the yield of one product
may have negative effect on the yield of others. Therefore, metabolic engineering
strategies should be considered to direct the material flow towards certain important
products. Alternatively, HPLW is a potential phosphate solubilizer, which can increase
the soluble phosphate concentration of an aqueous medium containing insoluble
inorganic phosphorus by 36 times. Therefore, it should be evaluated as a substitute of
PSB. As (i) PSB is a commercially successful biofertilizer, (ii) HPLW needs no
downstream processing for this application, (iii) it has no direct production cost involved,
and (iv) compared to PSB it is projected to have superior shelf life; the strategy has
considerable interest as additional revenue source for fermentative hydrogen
production. PHA and lipid production are other different indirect but attractive options for
sustainable utilization of HPLW. Methane can also be produced from HPLW; however,
this approach will consume all the byproducts of hydrogen production process which,
otherwise, would have better a market than methane. Moreover, methane production
needs further fermentation of HPLW; which is not the case for other byproducts.
Similarly, photo-fermentation and microbial electrolysis cell can be used for further
conversion of HPLW to hydrogen. However, high process cost and concern over its
scalability are the issues yet to be resolved. Overall, hydrogen biorefinery approach
carries tremendous industrial potential and hence, instead of concentrating on one
159
particular by-product, the entire process should be evaluated as an integrated strategy
and a proper cost benefit analysis of the approach is required.
Acknowledgments
CRIQ, NSERC, MRI (Quebec-Parana and Quebec-Vietnam) and INRS-ETE Canada
have been acknowledged for financial support. The authors are also thankful to “merit
scholarship program for foreign students (FQRNT)” for financial assistance to Mr.
Saurabh Jyoti Sarma.
160
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Table 2.3. 1: Summary of different reports on simultaneous hydrogen and ethanol production
SL.
Microorganism
Substrate
Process operation
No.
1
2
3
Hydrogen
Ethanol production
Reference
0.80 mol-H2/mol-
0.50 mol-ethanol/mol-
(Karadag et
glucose
glucose
al., 2010)
production
Mixed anaerobic
Glucose (9 g/L) and
culture from hot spring
additional nutrients
Microorganisms from
α-cellulose and
Batch study using 125
2.8 mmol-H2/g-
5.8 mmol-ethanol/g-
(Lin et al.,
cow dung sludge
xylose
mL serum bottles
cellulose and 0.3
cellulose and 1 mol-
2008)
mol-H2/mol-xylose
ethanol/mol-xylose
0.45L continuous flow
0.32 mol-H2/mol-
(Koskinen et
reactor
glucose
glucose
al., 2007)
Batch study using 165
2.49 mol H2/mol
30.6±2.4% carbons of
(You-Kwan
mL serum bottles
glucose
the
Oh et al.,
Enrichment microbial
Glucose/cellulose
cultures from hot
1L CSTR (Anaerobic)
spring
4
Citrobacter sp. Y19
Glucose
substrate
were
directed toward ethanol
2003)
production
5
Anaerobic
immobilized
sludge
Sucrose
on
Fluidized-bed reactor
0.64 mol-H2/mol-
0.24 mol-ethanol/ mol-
(Ken-Jer Wu
and packed-bed reactor
hexose
hexose
et al., 2007)
1.04 mol-H2/mol-
hexose
hexose
0.56 mol-H2/mol-
0.65 mol-ethanol/ mol-
polyethylene-octane
elastomer
Glucose
Fructose
174
SL.
Microorganism
Substrate
Process operation
No.
6
Hydrogen
Ethanol production
Reference
production
hexose
hexose
Enterobacter
Biodiesel industry
Two-compartment bio
0.77 mol-H2/mol-
(Sakai et al.,
aerogenes NBRC
effluent containing
electrochemical reactor
glycerol
glycerol
2007)
12010
glycerol
(liquid volume 300 mL x
(Zhao, 2009)
2)
7
Extreme thermophilic
Glucose (2g/L) and
Batch process using 250
1.58 mol-H2/mol-
microbial mixed
additional nutrients
mL serum bottles
glucose
glucose (equivalent to
culture
0.23g-ethanol/gglucose)
8
Enterobacter
Biodiesel industry
Batch process using 125
1.12 mol-H2/mol-
(Ito et al.,
aerogenes HU-101
effluent containing
mL serum bottles
glycerol
glycerol
2005)
56.6 ± 2.1 mmol /L
29.6 ± 0.5 mmol /L
(Sigurbjorns
glycerol
9
Newly isolated strain
Acid treated
Batch process using
(strain AK54) close to
cellulose
117.5 mL serum bottles
Thermo
hydrolysates
dottir et al.,
2012)
anaerobacterium
aciditolerans
10
Escherichia coli K12
Glycerol (20 g/L)
Study using 2.25 L
1.40 mmol /L
0.32 mmol /L
(Chaudhary
et al., 2011)
bioreactor
175
SL.
Microorganism
Substrate
Process operation
No.
11
Hydrogen
Ethanol production
Reference
11.72 mmol ethanol/h/L
(Han et al.,
production
Immobilized sludge
Molasses
Study using 12.5L CSTR
10.74 mmol H2/h/L
2011)
12
Clostridium sp. strain
Cellobiose (2 g/L)
URNW
Batch study using 27 mL
14.2 mmol-H2/L-
Balch tubes
medium
0.4 mmol-H2/L-medium
(Ramachand
ran et al.,
2011)
13
Mixed culture
Glucose
100–200 mL-H2/g-
operated reactor
glucose (25–40% H2)
Wastewater having
Expanded granular
3.47 mol-H2/mol-
62–128 mg-ethanol/L
(Guo et al.,
molasses
sludge bed
sucrose
(after 10 days)
2008)
Wastewater with
CSTR
0.45 L-H2/g-COD
Not specified
(Nanqi Ren
(maintained on
Not specified
(Hwang et
Semi-continuously
al., 2004)
wastewater sludge)
14
15
Mixed culture
Mixed culture
molasses
removed (35 days)
et al.,
2007b)
176
Table 2.3. 2: Ethanol production by microbial conversion of different organic substrates
SL.
Microorganism
Substrate
Process
Ethanol production
Reference
Baker’s yeast (Saccharomyces
Hydrothermal and
Batch process using
0.41 g/g (glucose)
(Kaparaju
cerevisiae)
enzymatically hydrolyzed
fermentation flask (200 mL)
No.
1
et al., 2009)
wheat straw
2
Saccharomyces cerevisiae
Marine macro algal
Batch process using Erlenmeyer
(Sacchorina latissima)
flask (100 mL)
0.45 % (v/v)
(Adams et
al., 2009)
biomass
3
Saccharomyces cerevisiae;
Processed tuber of
Zymomonas mobilis;
Jerusalem artichoke
flask (500 mL)
Rice straw hydrolysate
0.48 g/g
(Szambelan
et al., 2004)
Kluyveromyces fragilis
4
Pichia stipitis
0.45 g/g
al., 2009)
flask (250 mL)
5
Candida tropicalis
Rice straw hydrolysate
Batch process using
polycarbonate flask (150 mL)
177
(Huang et
0.48 g/g
(Oberoi et
al., 2010)
Table 2.3. 3: Summary of different reports on simultaneous hydrogen and 1, 3-propanediol production
Sl.
Microorganism
Substrate
Process
Hydrogen production
No.
1
1,3-propanediol
Reference
production
Mixed
culture
(heat-
Pure glycerol
treated)
Batch process
0.28 mol-H2/mol-glycerol
using serum
0.69 mol-PD/mol-
(Selembo et al.,
glycerol
2009)
bottles (500 mL)
2
Crude glycerol
0.59 mol-PD/molglycerol
3
4
Glucose
1.06 mol-H2/mol-glucose
No PD production
Microbial culture from
Biodiesel waste
Batch process
0.65 mol-PD/mol-
(Bingchuan Liu
organic soil
containing
using serum
(at shorter hydrogen
glycerol (at longer
et al., 2013)
glycerol
bottles (160 mL)
retention time)
hydrogen retention
time)
5
6
Halanaerobium
Batch process
0.39 mol-PD/mol-
(Kivistö et al.,
saccharolyticum
using serum
(without vitamin B12)
glycerol (with vitamin
2011)
(saccharolyticum)
bottles (25 mL)
Clostridium spp. from
Pure glycerol
Colombian soil
B12)
Batch process
0.1962 mol-H2/mol-
using bioreactor
glycerol
Not specified
(Bernal et al.,
2013)
(4 L)
7
Enterobacter
agglomerans
Pure glycerol
Batch process
Negligible H2 production
using 120 mL
178
0.51mol-PD/mol-
(Barbirato et
glycerol
al., 1995)
Sl.
Microorganism
Substrate
Process
Hydrogen production
No.
1,3-propanediol
Reference
production
penicillin flasks
8
Recombinant
Pure glycerol
Escherichia coli
Batch process
-
0.3 g-PD/g-glycerol
(Dabrowski et
al., 2012)
using 100 mL of
medium
9
Citrobacter freundii
Crude glycerol
Batch process
ATCC 8090
(20 g/L)
using 500 mL
-
4.85 g-PD/L-medium
(Ferreira et al.,
2012)
flasks
10
Clostridium butyricum
Crude glycerol
-
-
63.4 g-PD/L
et al., 2011)
CNCM 1211
11
Klebsiella pneumoniae
(Drożdżyńska
Pure glycerol
-
-
DA-1HB
179
70.5 g-PD/L
Table 2.3. 4: Summary of different reports on simultaneous hydrogen and butyric acid production
SL.
Microorganism
Substrate
Process
Hydrogen production
No.
1
Butyric acid
Reference
production
Clostridium
Microalgae
tyrobutyricum 3T
(Scenedesmus
-
1.36 mol-H2/mol-sugar
0.49
mol-butyric
(Ortigueira et
acid/mol-sugar
al., 2012)
1.85 mol-H2/mol-
0.27 g-butyric acid/g-
(Xiaoyi Wang
hexose
hexose
et al., 2009)
0.024 g-H2/g-glucose
0.42 g-butyrate /g-
(Xiaoguang
glucose
Liu et al.,
obliquus)
hydrolysate
2
Clostridium
butyricum
Molasses
W5
Batch
process
using
a
bioreactor with a working
volume of 1.5 L
3
Clostridium
Glucose
tyrobutyricum PAK-Em
5 L stirred-tank bioreactor
operated in fed-batch mode
2006)
4
Anaerobic fluidized bed
2.49 mol-H2/mol-
40.99 % of total
(de Amorim et
reactor used for the
reactor with a HRT of 2
glucose
soluble microbial
al., 2012)
treatment
hours
Anaerobic
sludge
of
of
Glucose 2 g/L
swine
products
wastewaters
5
Mixed culture from
Molasses
activated
3.1L continuous acidogenic
~ 40 %
720 mg/L
(NQ Ren et
al., 2007a)
reactor
sludge
6
Clostridium beijerinckii
Food waste
Batch H2 production using
180
231 mL H2/200 mL of
3,546 mg/L
(Jung Kon
SL.
Microorganism
Substrate
Process
Hydrogen production
No.
Butyric acid
Reference
production
KCTC 1785
500 mL serum bottle
Kim et al.,
medium
2008)
7
8
Sludge from anaerobic
Cassava
digester
wastewater
75 mL serum bottle
Sludge compost
Wastewater
Continuous fermentation
from sugar
340.19 mL-H2/ g-COD
36.24% of VFS
(Reungsang
produced
et al., 2007)
2.59 mol-H2/mol-
0.62 mol-butyrate/mol-
(Ueno et al.,
using a 5L vessel
hexose
hexose
1996)
2870 ± 60 mg/L
(Sang-Eun Oh
factory
9
Clostridium
Glucose (10 g
2.44 ± 0.16 mol-
acetobutylicum ATCC
COD/L)
using a 2L vessel
H2/mol-glucose
Glucose (25 g
~ 375 mL-H2/g-cell
COD/L)
150 mL serum bottle
Clostridium sp. AK14,
Glucose (20
isolated
mM)
23 mL serum bottle
Anaerobic digester
L-Arabinose
2.5 mol-H2/mol-
61.0 % of soluble
(Abreu et al.,
sludge enriched with
(30 g COD/L)
125 mL serum bottles
arabinose utilized
microbial products
2009)
et al., 2009)
824 (mutant strain M5)
10
11
Heat treated sludge
554.3 mg-COD/L
(Cheong et
al., 2006)
30.5 mM-H2
10.7 mM-butyrate
(Almarsdottir
et al., 2010)
from hot spring
12
supplementary fat
181
SL.
Microorganism
Substrate
Process
Hydrogen production
No.
13
Butyric acid
Reference
production
Anaerobically digested
Corn syrup
A novel continuous
sludge from
waste
fermentation process using
wastewater treatment
3.2 mol-H2/mol-hexose
3793 ± 671 mg-
(Hafez et al.,
butyrate/L
2009)
0.36 mol -
(Kurokawa et
butyrate/mol-glucose
al., 2005)
9.3 mmol- butyrate/L-
(Yasuda et al.,
culture/ h
2010)
131.7 mg-butyrate/g-
(Young Joon
sucrose
Lee et al.,
a 5L bioreactor
plant
14
Enterobacter
aerogenes st.
Glucose (10
Batch process using a 2.5L
g/L)
bioreactor
Starch
~15 mmol/g-cell/h
E. 82005
15
Clostridium sp HN001
2.0 mol-H2/mol-hexose
using a 1L bioreactor
operated at HRT of 9h
16
Seed sludge from
Sucrose (10
Batch process using glass
laboratory scale
g/L)
bottles
anaerobic digester
131.9 mL-H2/g-sucrose
2001)
182
Table 2.3. 5: Bio-methane production using dark fermentative hydrogen production liquid waste
Sl.
Original
No.
substrate
1
2
Process
Microorganism/inoculum
Potato
Continuous
two
processing
process using one 1L and
waste
one 5L bioreactor
Food waste
Continuous
two
stage
H2 production
CH4 production
Sludge
Digested sludge
Hydrogen
Methane
production
production
Reference
119 mL-biogas/h
141 mL-biogas/h
(Zhu et al.,
with 45% (v/v) H2
with 76% (v/v)
2008)
CH4
stage
process using one 10L
Sludge from anaerobic digester of
205 mL-H2/g-
464 mL-CH4/g-
(Chu et al.,
wastewater treatment plant
volatile solid
volatile solid
2008)
Sludge
Digested sludge
1.62 ± 0.2 mol-
323 mL-CH4/g-
(Kyazze et
H2/mol-hexose
COD
al., 2007)
11.1 L-H2/L-fed/d
47.4 L-CH4/L-
(Dong-Yeol
fed/d
Lee et al.,
(H2 production) and one
40L
(CH4
production)
bioreactor
3
Sucrose
Continuous
two
stage
from
mesophilic
supplemented
CSTR
digester
with sucrose
(H2
production)
and one 20L upflow filter
(CH4 production)
4
Food waste
Continuous
two
stage
Heat
treated
from
sludge
(H2 production) and one
anaerobic
digester (without
40L
digester
treatment)
(CH4
production)
from
Sludge
anaerobic
bioreactor
183
2010)
Sl.
Original
No.
substrate
5
Process
Microorganism/inoculum
Partially
Continuous
processed
garbage
and
paper waste
CSTR
(H2
two
stage
production)
and one 500L internal
recirculation packed-bed
H2 production
CH4 production
Thermophilic
Anaerobic sludge
microbial
consortium
from activated
sludge compost
from
Hydrogen
Methane
production
production
5.4 L-H2/L/d
6.1 L-CH4/L/d
Reference
(Ueno et
al., 2007)
a
thermophilic
methanogenic
process
reactor (CH4 production)
6
Cassava stillage
Continuous two stage H2
Anaerobic granular sludge
and CH4 production using
300 mL reactors
73.7 ± 3.2 mL-
379.6 ± 13.0 mL-
(Wen Wang
H2/g-volatile solid
CH4/g-volatile
et al., 2011)
solid
184
Figure 2.3. 1: Possible applications of the liquid waste from dark fermentative hydrogen
production.
185
1800
350
1600
Concentration
1400
Price
1200
300
250
1000
200
800
150
600
100
400
50
200
0
0
Price (analytical grade) in $/L (or $/kg)
Byproduct present in HPLW (mg/L)
Figure 2.3. 2: Reported concentration levels of different metabolites present in HPLW and their
market price in US dollars (Han et al., 2010, Lalaurette et al., 2009, Selembo et al., 2009).
186
Methane
Ethanol
PD
Butyric
Propionic
Succnic
acid Acetic acid acid Lactic acid acid
valerate
Free energy of formation (KJ/mol)
0
-100
-200
-300
-400
-500
-600
-700
-800
Figure 2.3. 3: Free energy of formation (25º C) of different metabolites produced during
biohydrogen production.
187
PART-IV
BIO-HYDROGEN PRODUCTION BY BIODIESEL DERIVED CRUDE
GLYCEROL BIOCONVERSION: A TECHNO-ECONOMIC EVALUATION
Saurabh Jyoti Sarmaa, Satinder Kaur Brara, Yann Le Bihanb, Gerardo Buelnab
a
INRS-ETE, 490, Rue de la Couronne, Québec (QC), Canada G1K 9A9
b
Centre de recherche industrielle du Québec, Québec(QC), Canada G1P 4C7
[email protected])
Bioprocess and Biosystems Engineering (2013) 36 (1): 1-10
189
Résumé
La production mondiale de biodiesel est en constante augmentation et il est
proportionnellement accompagné d'une énorme quantité de glycérol brut (GB) comme
sous-produit. En raison de sa nature brute, le GB a un intérêt commercial très bas, bien
que son homologue pur a des applications industrielles. En variante, le GB est une très
bonne source de carbone et peut être utilisé comme matière première pour la
production d'hydrogène par fermentation. En outre, son utilisation présente un double
avantage, à savoir qu'elle offre une méthode durable pour l'élimination des déchets de
fabrication du biodiesel, et qu'elle produit des biocarburants à faible émission de gaz à
effet de serre (GES). La fermentation en deux étapes, comprenant la phase sombre et
la photo-fermentation est une des options les plus prometteuses pour la production de
biohydrogène. Dans la présente étude, la faisabilité technico-économique d'un tel
processus en deux étapes a été évaluée. L'analyse a été faite sur la base des avancées
récentes dans la production d'hydrogène par fermentation à l'aide du GB comme
matière première. L'étude a été réalisée avec une référence particulière sur le marché
du biodiesel en Amérique du Nord et, plus précisément, les données disponibles pour
les provinces canadiennes et le Québec ont été utilisées. Sur la base de notre analyse
technico-économique, l'augmentation du coût de production a été démontrée comme
étant le principal goulot d'étranglement dans la production commerciale de l'hydrogène
par fermentation. Cependant, certaines options alternatives réalisables pour la
réduction des coûts de procédé ont été identifiées. En plus des réductions des
émissions de GES, le processus de bioconversion du GB dans son ensemble offre des
avantages environnementaux supplémentaires. Les estimations calculées démontrent
que la bioconversion d'un kg de glycérol brut (70% p/v) permet de réduire les émissions
de GES de 7,66 kg d'équivalent CO2.
Mots clés: biodiesel; biohydrogène; glycérol brut; gaz à effet de serre; l'évaluation
technico-économique
190
Abstract
Global biodiesel production is continuously increasing and it is proportionally
accompanied by a huge amount of crude glycerol (CG) as by-product. Due to its crude
nature, CG has very less commercial interest; although it’s pure counterpart has
different industrial applications. Alternatively, CG is a very good carbon source and can
be used as a feedstock for fermentative hydrogen production. Further, a move of this
kind has dual benefits, namely it offers a sustainable method for disposal of biodiesel
manufacturing waste as well as produces biofuels and contributes in greenhouse gas
(GHG) reduction. Two-stage fermentation, comprising dark and photo-fermentation is
one of the most promising options available for bio-hydrogen production. In the present
study, techno-economic feasibility of such a two-stage process has been evaluated. The
analysis has been made based on the recent advances in fermentative hydrogen
production using CG as a feedstock. The study has been carried out with special
reference to North American biodiesel market; and more specifically, data available for
Canadian province, Québec City have been used. Based on our techno-economic
analysis, higher production cost was found to be the major bottleneck in commercial
production of fermentative hydrogen. However, certain achievable alternative options for
reduction of process cost have been identified. Further, the process was found to be
capable in reducing GHG emissions. Bioconversion of one Kg of crude glycerol (70 %
w/v) was found to reduce 7.66 Kg CO2 eq (equivalent) GHG emission, and the process
also offers additional environmental benefits.
Key words: Biodiesel; bio-hydrogen; crude glycerol; greenhouse gas; techno-economic
evaluation
191
Introduction
Biodiesel is one of the renewable alternatives of fossil fuel which is mainly produced
from vegetable oils and animal fat. Due to availability of feedstock and requirement of
very simple technology for its production and its role in greenhouse gas (GHG)
reduction; global production of biodiesel is rapidly increasing. European Union is the
largest producer of biodiesel in the world followed by North America (NA). NA’s
biodiesel market is mainly dominated by the U.S., which is the second largest biodiesel
producing nation with production of nearly 17.7 percent of the world's biodiesel in 2009.
Further, biodiesel production in US has been promoted by US government through
various subsidies and policies. The revised renewable fuel standard (RFS2) program
has been devised, according to which by 2012 annually a minimum of 1 billion gallons
of biodiesel is to be blended into U.S. diesel fuel and it will require approximately 36
billion
gallons
of
renewable
fuel
by
2022
(http://www.biodieselmagazine.com/articles/4080/report-12-billion-gallons-of-biodieselby-2020. Aaccessed 27 Dec 2011). Similarly, Canada is a major biodiesel producer in
NA and Canadian government has set a target of 17 percent reduction of GHG by 2020
and
2
%
renewable
content
will
be
mandatory
for
diesel
oil
(http://www.ec.gc.ca/default.asp?lang=En&n=714D9AAE-1&news=BDE26528-CBCB4008-949D-CDFDFBD054FB. Aaccessed 27 Dec 2011; Regulations amending the
renewable fuels regulations, Canadian environmental protection act, 1999, department
of
the
environment,
Canada.
https://tsapps.nist.gov/notifyus/docs/wto_country/CAN/corrigenda/pdf/CAN311_add_3(e
nglish).pdf. Aaccessed 27 Dec 2011). Hence, biodiesel production in NA will be
expected to increase significantly in the coming decade. It is well established that
biodiesel production is accompanied by generation of glycerol as a by-product, by
weight, which is almost 10 % of the total biodiesel produced (Thompson et al., 2006).
Therefore, there will be a sharp increase in crude glycerol (CG) production in recent
future and management of such a huge amount of waste will be a problem for biodiesel
manufacturers. Interestingly, CG can be used as a feedstock for production of number
of valuable products; however, due to presence of various impurities in biodiesel
derived glycerol, product recovery is expensive (Pachauri et al., 2006). Alternatively, CG
192
can be used as a substrate for hydrogen production, in which case, produced H2 is
automatically separated from the media and it can be recovered using simple
technology such as, pressure swing adsorption. Moreover, produced H2 can be used as
a fuel with no harmful emissions during its combustion (Auer et al., 2000); and hence, it
has the potential of reducing GHG emission by replacing conventional fossil fuels.
Further, the microbial biomass generated during H2 production process can be used for
biogas production by anaerobic digestion. There are number of different strategies for
H2 production that have been investigated (Das et al., 2001, Hawkes et al., 2007, Nath
et al., 2004); however, based on comparison of different processes, very high H2
production potential has been reported with a two-stage fermentation process. In an
investigation by Yokoi et al (2002), dark fermentation was shown to produce 2.7 mol H2
mol-1 glucose and subsequent photo-fermentation of spent media by Rhodobacter sp.
M-19 was reported to produce additional 4.5 mol H2 mol-1 glucose (Manish et al., 2008,
Yokoi et al., 2002). Further, by production of 1 Kg of hydrogen, such two-stage process
has the potential to reduce GHG emissions by 7.31–9.37 Kg CO2 eq (Manish et al.,
2008). Therefore, the purpose of this article is to make a techno-economic evaluation of
a two-stage fermentation process for H2 production by CG bioconversion based on
different assumptions corresponding to the recent technological progresses and
available literature data.
Biodiesel and CG production in NA
Considering global biodiesel market, US and Canada are the two major representatives
of North America (NA). Therefore, for convenience of estimation, data available for US
and Canada together will be considered as the data for whole NA. Soybean oil, canola
oil and animal fat (tallow) are common feedstock used for biodiesel production in NA.
Total annual production of soybean oil in NA is estimated to be 8.84 million metric tonne
(http://www.soystats.com/2011/page_21.htm.
Aaccessed
27
Dec
2011;
http://www.indexmundi.com/agriculture/?country=ca&commodity=soybeanoil&graph=production. Aaccessed 27 Dec 2011). Tallow is the second major biodiesel
feedstock
in
NA
and
its
annual
production
is
2.73
million
metric
tonne
(http://www.petfoodindustry.com/Columns/Ingredient_Issues/2390.html Aaccessed 27
193
Dec 2011; C.B. Prakash, A critical review of biodiesel as a transportation fuel in
Canada.
A
technical
report.
http://www.dieselduck.ca/library/05%20environmental/1998%20Biodiesel%20in%20Can
ada.pdf. Aaccessed 27 Dec 2011). Similarly, Canada is the topmost canola oil producer
in the world and annual production of canola oil in NA is 2.06 million metric tonne
(http://www.canolacouncil.org/ind_overview.aspx. Aaccessed 27 Dec 2011; The CRB
Commodity
Yearbook
2007.
By
Commodity
Research
Bureau.
http://books.google.ca/books?id=YqKVT3w67KoC&pg=PA317&lpg=PA317&dq=The+C
RB+Commodity+Yearbook+2007&source=bl&ots=sbgW7MZjEj&sig=rZlGYVgWr8zce3
M8YDZ45CmuDAo&hl=en&sa=X&ei=JJH7ToSwM8ru0gG9xsC5Ag&ved=0CDkQ6AEw
Ag#v=onepage&q=The%20CRB%20Commodity%20Yearbook%202007&f=false.
Aaccessed 27 Dec 2011). Currently, in US alone, there are nearly 170 biodiesel
manufacturing plants (Glycerin becomes “The Newest Biofuel” with the MK Glycerin
Burner from AlterHeat. http://www.glycerinburners.com. Aaccessed 27 Dec 2011) and
annually they are producing around 2839.05 million liters of biodiesel (http://www.earthpolicy.org/datacenter/xls/book_wote_ch9_biofuels_9.xls. Aaccessed 27 Dec 2011).
Annual production of biodiesel and biodiesel feedstock in leading North American
nations are summarized in Table 2.4.1 and by weight, the total annual biodiesel
production in NA is found to be only 19.51 % of the available feedstock. Therefore, NA
has the potential to further increase its annual biodiesel production. As mentioned
earlier, during biodiesel production, a huge amount of CG is produced as waste byproduct and annually around 0.2 million metric tonne of CG have been produced in NA
(http://www.biodieselmagazine.com/articles/1123/combating-the-glycerin-glut.
Aaccessed 27 Dec 2011). Considering annual production of excess feedstock and
favourable government policy for increased biodiesel production and well established
market, it can be expected that in the coming years, North American biodiesel
production will eventually increase. Therefore, in near future, CG production will also be
further increased and a sustainable method of CG management will be necessary.
194
CG bioconversion process overview
Till date, various strategies have been applied for H2 production by CG bioconversion
among which dark anaerobic fermentation is the most well studied process. Ngo et al
(2011) have reported the production of 2.73 ± 0.14 mol-H2 mol-1 glycerol by Thermotoga
neapolitana (Ngo et al., 2011). Similarly, Fernandes et al. (2010) have reported the
production of 200 ml-H2 g-1 COD (Fernandes et al., 2010). In another similar
investigation, Marques et al (2009) have used Enrerobacter aerogenes and the authors
have reported the production of 2.5 dm3-H2 dm-3 medium (Marques et al., 2009).
However, in all such cases despite very high hydrogen yield, bioconversion efficiency is
still limited by accumulation of fermentation end products. Alternatively, hydrogen
production efficiency of a two-stage process is reported to be highest among other
microbiological processes (Manish et al., 2008). In this case, end-products accumulated
during first phase of fermentation are further utilized by subsequent fermentation.
Therefore, in this study we will investigate the techno-economic feasibility of a two-stage
process, where dark fermentation will be followed by photo-fermentation for maximum
bioconversion of CG. Figure 2.4.1 represents the proposed process and bioconversion
of one Kg of CG will be evaluated considering the cost of materials and power
requirement in various steps.
In general, CG from biodiesel manufacturing plant contains approximately 70 % (w/w)
glycerol (Sabourin-Provost et al., 2009, Priscilla A. Selembo et al., 2009b, Tang et al.,
2009). For most bioconversion study, around 0.25 % to 1 % (w/v) glycerol has been
successfully utilized for bio-hydrogen production (Ito et al., 2005, Kivisto et al., 2010,
Ngo et al., 2011, P. A. Selembo et al., 2009a). However, high H2 yield has been
reported at relatively low initial glycerol concentration (Ito et al., 2005). Therefore, in this
study 0.25 % (w/v) glycerol will be considered as initial glycerol concentration to be
used for H2 production. Hence, for bioconversion of one kilogram of CG, it should be
diluted to 400 L (0.25 % w/v) solution. Therefore, a 1000 L continuous stirred tank
reactor (CSTR) will be considered for entire bioconversion process and different
calculations will be based on this assumption.
195
Inoculum preparation
5% (v/v) inoculum will be considered for proposed bioconversion study and hence, 20 L
inoculum will be required for 400 L (20 L + 380 L) media. It will be developed in a 50 L
CSTR containing 20 L MD media. The MD medium contains per liter of deionized water:
glucose monohydrate (5 g), casein peptone (5 g), yeast extract (0.5 g), KH2PO4 (2 g),
MgSO4.7H2O (0.5 g) and L-cysteine hydrochloride (0.5 g) (Thonart et al., 2010). Based
on the catalogue (2011) of a professional chemical supplier (VWR, Canada); total cost
for 20 L media component will be $44.88. Similarly, electricity charges will be applicable
for sterilization of media and incubation of inoculum at 37±1ºC. It will take approximately
2 h to sterilize the total media and approximately 15 h of incubation for inoculum
development. For a production scale bioreactor, power input requirement is around 1-3
kW/m3
(Scale-up
of
bioprocess,
Chapter
11
in
NV&L.
http://www.chemeng.lth.se/kte071/Arkiv/scale-up.pdf. Aaccessed 27 Dec 2011). Hence,
considering a 50 L CSTR having in situ sterilization facility and with 0.05 kW
requirements, 0.85 kWh of power will be needed for entire inoculum development
process. According to the present electricity tariff of Hydro-Quebec, which is 4.46 cent/
kWh;
electricity
cost
for
inoculum
development
will
be
$
0.037
(http://www.hydroquebec.com/residential/tarif-affaires.html. Aaccessed 27 Dec 2011).
CG pre-treatment
It is well known that CG from biodiesel manufacturing plant contains different impurities.
Methanol and soap are the two major impurities present in CG and they may be
inhibitory to microbial growth (Athalye et al., 2009, Chi et al., 2007, Ngo et al., 2011).
However, methanol evaporates at 65 ±1 °C; hence, the sterilization of CG is capable of
removing significant amount of methanol (Ethier et al., 2011, Pyle et al., 2008).
Similarly, the other major impurity present in CG is soap and it can be removed by
precipitation through pH adjustment (Athalye et al., 2009, Ethier et al., 2011). As
suggested by Chi et al. (2007), to reduce the viscosity, CG will be first diluted with
distilled water at 1:4 ratio. Later, HCl will be added to reduce the pH to 6.5 to convert
the soluble soap into insoluble free fatty acids and precipitated solid will be discarded
(Chi et al., 2007). In general, initial pH of CG is around 11-12 and after dilution with
196
distilled water, total volume of the CG solution will be around 5L. Hence, at least 200 ml
of HCl will be required for its pH adjustment and it will cost approximately $ 2.38.
Media preparation
It can be found from the literature that when CG is diluted with only deionized water, no
significant microbial growth and hydrogen production was observed (Ito et al., 2005).
However, after dilution with suitable buffer solution containing supplementary nutrient,
significant improvement in cell growth and hydrogen production has been observed (Ito
et al., 2005). Similarly, Selembo et al (2009) and some other workers have also used a
suitable nutrient solution for CG bioconversion experiment (Fernandes et al., 2010,
Sabourin-Provost et al., 2009, P. A. Selembo et al., 2009a). Therefore, for
bioconversion, CG will be diluted with phosphate buffer containing 4.58 g/L Na2HPO4
and 2.45 g/L NaH2 PO4. H2O (pH 7.0) (P. A. Selembo et al., 2009a). Further, addition of
yeast extract was found to increase the CG consumption rate (Ito et al., 2005); hence
1g/L yeast extract will be added to the media containing CG. For 380 L media, about
1740.4 g Na2HPO4, 931 g NaH2 PO4.H2O and 380 g yeast extract will be required. The
cost for these items will be $ 96.97, $ 38.82 and $ 25.25, respectively.
Anaerobic dark fermentation
Prepared media will be sparged with N2 for 5 to 10 min followed by sterilization at
121±1oC for 20 min. A nitrogen generator will be used for obtaining pure N2 flow and the
reactor vessel along with the media will be sterilized in situ. Ito et al (2005) have
reported complete consumption of glycerol within one day in a batch experiment for
initial concentration as high as 2.5 % (Ito et al., 2005). Further, Selembo et al (2009)
have observed nearly 18 h of lag phase in H2 production when CG bioconversion was
investigated using mixed microbial culture (P. A. Selembo et al., 2009a). However, they
have shown that by using a freshly collected inoculum from previous CG fermentation,
the lag phase of H2 production could be reduced to 5 h. Similarly, Ngo et al (2011) have
shown that when Thermotoga neapolitana was used as inoculum, H2 production rapidly
increased after 6 h of incubation. However, after 24 h of cultivation, the process pH
dropped and the rate of H2 production was also declined (Ngo et al., 2011). From these
observations, it can be assumed that a batch of H2 production process using CG as
197
feedstock will be completed within 48 h. Further, Mahanty et al (2010) have shown that
charge of electricity for a 3 L bioreactor operated at constant agitation and aeration was
$ 0.38 per hour, however, nearly half of the cost was due to aeration (Mahanty et al.,
2010). In the case of anaerobic bioconversion, aeration will not be required and the
charges will be even lower. As it is mentioned earlier, maximum electricity requirement
of a 1000 L CSTR is 1 kW, hence, electricity charge for entire dark fermentation can be
calculated to be around $ 2.14.
Photo fermentation
For 400 L spent media of dark fermentation; 20 L inoculum (5% v/v) of phototrophic
bacteria will be used. Hence, cost of inoculum development can be assumed to be the
same as compared to that of dark fermentation. For photo- fermentation, pH adjustment
of the spent media and addition of supplementary media component may be necessary
(Tao et al., 2007). Therefore, we will assume that 0.5 g/L yeast extract and 200 g NaOH
will be added to the spent media before sterilization. It is known from the literature that
during photo-fermentation, after 6th day, the rate of H2 production decreased (SabourinProvost et al., 2009, Tao et al., 2007). In this investigation, we will assume that
fermentation will be stopped at 144 h. Usually, 4000 to 5000 lux light intensity is used
for photo-fermentation (Tao et al., 2007, Yokoi et al., 2002). Sabourin and Hallenbeck
(2009) have used a 50 W halogen light for a 125 ml reactor used for CG bioconversion
and reported very high hydrogen yield (Sabourin-Provost et al., 2009). In the present
market, 50 L photo-bioreactor (Applicon ®, Netherlands) are available with 120 watt
lighting panel (http://www.applikon-bio.com. Aaccessed 27 Dec 2011). Hence, for
convenience of calculation, a 1000 L photo-bioreactor with 2400 W lighting panel will be
considered for bioconversion of 400 L spent media and electricity charges for the entire
operation will be $21.83.
Treatment of waste and biogas production
Based on the estimation made so far, nearly 400 L of waste will be generated during H2
production from one Kg of CG. Considering the volume, possible microbial biomass and
nutrient content of CG bioconversion waste, it can be easily concluded that a proper
treatment will be necessary before it can be released to the environment. In a recent
198
report, Manish and Banerjee (2008) have concluded that biological hydrogen production
is very efficient only when recovery and utilization of by-products is considered (Manish
et al., 2008). Therefore, the waste will be further digested in an anaerobic digester for
biogas generation. Considering the media composition of bioconversion experiments,
the spent media should contain approximately 5.52 Kg dry biomass. Siswo (2010) have
reported that by anaerobic digestion of effluent, 726.43 ml biogas /gram total solid can
be produced (Siswo, 2010). Hence, nearly 4010.9 L biogas can be produced using the
spent media. Further, 1 m3 (1000 L) biogas is equivalent to 0.6 L of diesel oil (Michael
AM,
Khepar
SD,
Sondhi
SK.
Water
wells
and
pumps.
http://www.mcgrawhill.ca/professional/products/9780071591201/water+wells+and+pum
ps. Aaccessed 27 Dec 2011). Therefore, produced biogas can replace 2.40 L fossil
diesel of around $ 3.27 (136.2 cents/ L).
Building construction, equipment & labour
Following equipment will be required for bioconversion of 1 Kg of CG: N2 generator, 50
L CSTR, 1000 L CSTR, 1000 L photo-bioreactor, pressure swing adsorption technology
(for H2 separation) and anaerobic digester. In general, instruments have 3-4 years
warranty and remain in good condition for 10 years with minimum maintenance and all
buildings have at least 30 years of lifespan (Lardon et al., 2009). Based on such facts,
for present estimation it will be assumed that cost of construction, equipment and labour
add 10 % additional expenses in H2 production process.
Hydrogen yield
Still only a few reports are available on H2 production by CG bioconversion and
maximum H2 yield reported in all such available reports are summarized in Table 2.4.3.
Dark fermentation with pure and mixed culture, photo-fermentation as well as microbial
electrolysis cell has been used so far for H2 production from CG. Culture conditions and
experiment setups in all such methods are different from each other and hence,
maximum H2 yield in all such cases are different. From Table 2.4.3 it can be seen that
Sabourin-Provost (2009) have reported that a maximum yield of 6 moles of H2 per mole
glycerol has been achieved, which is 75 % of maximum possible theoretical yield
(Sabourin-Provost et al., 2009). Hence, based on molecular weight, from 92.09 g
199
glycerol, maximum 6 g H2 could be generated. Therefore, from one Kg of CG (70 %
w/v), using presently available technology, approximately 45.6 g of H2 could be
generated.
A cost benefit analysis of CG bioconversion process
Table 2.4.2 and Figure 2.4.2 represent the estimated cost distribution of CG (1 Kg)
bioconversion process. From this analysis, it can be summarized that the operation cost
of the processes considered in this study is very high. Similarly, different CG
bioconversion and bio-hydrogen production processes till now followed by different
researchers are more or less identical to the process assumed here and hence, they
are also not economically feasible for commercial application due to very high process
cost involved. However, these processes offer number of different options for reduction
of the process cost. Firstly, from Figure 2.4.2, it is evident that cost of media
constituents is about 82 % of the total process cost. It is only because of the large
volume of nutrient media that has to be added to dilute the CG to a very low initial
concentration for its bioconversion. This problem can be easily solved if the experiment
can be carried out at higher initial CG concentration, so that, total volume of
supplementary media needed for the process can be reduced. In the present
estimation, initial concentration of CG was considered to be 2.5 g/L; however, numbers
of reports are available, where H2 production was demonstrated at 10 g/L or higher
initial concentrations (Kivisto et al., 2010, Ngo et al., 2011, P. A. Selembo et al., 2009a).
Even report on bioconversion of CG at 50 g/L initial concentration is also available (Liu
et al., 2011). Therefore, 40 times or more reduction in process cost will be possible if
bioconversion process is carried out at higher initial CG concentration. However, at high
initial substrate concentration, H2 production rate will be decreased and it will take
longer incubation time to complete a batch of bioconversion reaction. Interestingly, from
Figure 2.4.2, it is clear that cost of electricity is only 8% of the total process cost
(Quebec City, Canada scenario). Therefore, a longer incubation time will not have much
effect on the total process cost. Secondly, the cost of inoculum development is nearly
27 % of the total production cost. However, if the residual biomass of previous batch is
used as inoculum, the process cost can be further reduced by 27 %. Similarly, without
200
any supplementary material, CG alone can also support microbial growth and it can be
fermented for H2 production. In that case, more than 92 % reduction in process cost is
possible; although, there is a possibility of reduction in H2 yield. However, before ruling
out the possibility of fermentation of CG without any supplementary media, maximum H2
yield of such process should be experimentally determined and its economic feasibility
should be evaluated. Further, cheap nitrogen sources such as, slaughter house
wastewater; brewery wastewater as well as activated sludge from wastewater treatment
plant should also be evaluated as supplementary nutrient for CG bioconversion
process. This option has the potential of maintaining high H2 yield as well as reduction
of process cost. From Figure 2.4.3 it can be seen that the electricity requirement of
photo-fermentation is more than 90 % of total electricity requirement of the process. It is
mainly because of the high intensity light source which needs to be maintained
throughout the fermentation period. Interestingly, from Table 2.4.2 and Figure 2.4.3, it
can be concluded that the electricity cost for dark fermentation is nearly 10 times lesser
than that of photo-fermentation. Therefore, instead of two-stage dark and photofermentation, repeated batch dark fermentation may be a suitable option, where one
cycle of dark fermentation will be followed by another cycle of dark fermentation at
different environmental conditions. For example, spent media (containing residual
glycerol) of first dark fermentation can be diluted with fresh media before second cycle
of dark fermentation; pH, temperature and microorganism used for first cycle may be
different in second cycle. Such an approach may be helpful in maximum utilization of
glycerol and increasing H2 yield per mole glycerol utilized; and also will cut the
additional electricity cost of photo-fermentation. However, it is only an assumption and it
needs to be established by further investigation.
One
Kg
of
mass
of
hydrogen
has
an
energy
value
of
120-142
MJ
(http://hypertextbook.com/facts/2005/MichelleFung.shtml. Aaccessed 27 Dec 2011).
Alternatively,
energy
density
for
diesel
fuel
ranges
(http://hypertextbook.com/facts/2006/TatyanaNektalova.shtml.
from
32
to
40
MJ/L
Aaccessed
27
Dec
2011). Therefore, 1 Kg of H2 can replace 3.55 L of conventional diesel. Similarly, it has
been already mentioned that using presently available technology, by bioconversion of
1 Kg of CG, 45.6g H2 and 4010.9 L biogas can be obtained. Considering the energy
201
content of the two fuels, hydrogen and biogas, biofuels produced from 1 Kg of CG are
capable of replacing 2.56 L of fossil diesel. Hence, 0.2 million metric tonne CG annually
produced in NA can replace 512 million liters of fossil diesel worth of 697.34 million
dollar (Table 2.4.5).
Estimation of environmental benefit of CG bioconversion process
GHG emission reduction
Methane yield coefficient of crude glycerol is 0.306 m3 CH4/Kg glycerol (Siles Lopez et
al., 2009). Therefore, if the total CG produced in NA is disposed to ultimate landfill,
annually at least 61.2 million m3 CH4 will be generated. Alternatively, if CG is converted
to animal feed, it will generate 659 Kg CO2eq/tonne crude glycerol (Carbon Life Cycle
Calculation
for
Biodiesel,
Home
Grown
Cereals
Authority,
United
Kingdom.
http://www.co2star.eu/final-documents/Annex3-4-WP2D3_Life%20cycle%20biodiesel.pdf. Aaccessed 27 Dec 2011). In such a situation, where
very few literature data on CG GHG potential are available, for present estimation,
these figures will be assumed as CG GHG emission potential. Further, during
production (refining) of 1 Kg fossil diesel approximately 360.41 g CO2 is released to the
atmosphere; therefore, production of 2.56 L diesel will generate 0.77 Kg CO2 eq GHG
(Sheehan et al., 1998). From Table 2.4.2, total electricity requirement for bioconversion
of 1 Kg of CG can be estimated to be 539.3 kWh. According to a recent report by
Environment Canada, CO2 emission coefficient for electrical power (Quebec, Canada,
2008)
is
2
g
CO2
equivalent
per
kWh
(http://www.ec.gc.ca/ges-
ghg/default.asp?lang=En&n=EAF0E96A-1. Aaccessed 27 Dec 2011). Therefore, during
bioconversion process of 1 Kg of CG, nearly 1.07 Kg of CO2 will be generated. This
estimation will be valid only when it is considered that CG produced in a biodiesel
manufacturing plant is used in the same plant for H2 production and GHG emission due
to CG transportation is negligible. As a fuel, liquid H2 produces very low or zero
emissions and the only waste product while burning hydrogen is water vapour, with a
small amount of NOx (Nojoumi et al., 2009). Similarly, during combustion of 1 Kg
biogas, 748.02 g GHG (CO2 and CH4) is generated (Yu et al., 2008). Therefore, few
grams of biogas produced during bioconversion of 1 Kg CG will have a very negligible
202
contribution on GHG emission, and hence, it is not considered for present estimation.
Similar to economic cost benefit analysis, GHG emission due to CG bioconversion plant
construction is assumed to be 10 % of the same of CG bioconversion process. From
previous discussion it can be found that H2 and biogas produced during bioconversion
of 1Kg CG is capable of replacing 2.56 L of diesel. Further, combustion of one liter
diesel
produced
around
2.9
Kg
CO2
(http://www.abc.net.au/tv/carboncops/factsheets/cc_cars.pdf. Aaccessed 27 Dec 2011).
Therefore, as shown in Table 2.4.4, biofuels produced during bioconversion of 1 Kg of
CG has the potential to reduce nearly 7.42 Kg CO2eq of GHG by replacing the fossil
fuel. However, supplementary media components needed to be used in both dark and
photo-fermentation will also make a small contribution towards GHG emission.
Therefore, net GHG reduction potential of the present process will be slightly lower than
the estimated value. However, if any industrial waste could be used as supplementary
material, as previously discussed, GHG emission reduction potential of the proposed
process will further increase. Thus, overall environmental and economic benefit of the
proposed process is summarized in Table 2.4.5.
An outlook on other environmental benefits
Crude glycerol is an environmental hazard and it has objectionable effect on human
health. According to material safety data sheet (MSDS) of CG, it can enter the body by
inhalation, ingestion or through skin and may cause irritation in respiratory track, skin
and eye. Ingestion may cause diarrhea, nausea and headache and chronic exposure
may cause kidney injury. Persons suffering from skin and eye problems and with liver
and kidney disorder are more prone to the adverse effect CG (Material safety data
sheet
(crude
glycerol):
http://www.biodieselgear.com/documentation/MSDS_Glycerol.pdf. Aaccessed 27 Dec
2011). According to US-EPA, land filling, composting, land application, subsurface
disposal, direct discharge to surface water (dumping), treatment at a publicly owned
treatment works (POTW) and anaerobic digestion are the different options of CG
management (Environmental laws applicable to construction and operation of biodiesel
production
facilities,
U.S.
Environmental
Protection
Agency.
http://www.epa.gov/region07/priorities/agriculture/pdf/ethanol_plants_manual.pdf.
203
Aaccessed 27 Dec 2011). However, all such methods have certain limitations. Firstly,
due to the presence of methanol, CG may have a flash point well below 140º F, which
will make it an ignitable hazardous waste. If the particular crude glycerin waste is
considered as a hazardous waste, for landfill disposal it must meet land disposal
restriction universal treatment standards of 40 CFR (Code of Federal Regulations)
268.48 and it will further increase biodiesel waste disposal cost. Additionally, some
landfills refuge CG due to the concern of spontaneous combustion. Secondly, CG is
purely a carbon source and cannot be composted alone and it requires a source of
nutrient. Also, because of very high BOD, high energy content, and viscosity of CG,
vigorous management of the compost would be required to maintain its aerobic nature
(Environmental laws applicable to construction and operation of biodiesel production
facilities,
U.S.
Environmental
Protection
Agency.
http://www.epa.gov/region07/priorities/agriculture/pdf/ethanol_plants_manual.pdf.
Aaccessed 27 Dec 2011). Additionally, pH of CG is very high and it has little buffering
capacity [54]; hence, during composting, pH should be externally maintained in
composting range. Similarly, either due to high salt content, high methanol content or
limited transpiration due to physical barrier posed by viscous CG, direct land application
can potentially burn the plant cover of the particular land. Further, due to high BOD, CG
may be harmful to aquatic organisms and its direct discharge to surface water is
prohibited.
Alternatively, presently proposed system of bioconversion of CG to produce hydrogen
and anaerobic digestion of the biomass obtained from the process may be a substitute
of these different CG treatment methods. Therefore, from environmental point of view,
the present method has dual benefit of reducing environmental pollution by proper
disposal of CG as well as potential contribution towards GHG reduction by production
renewable and eco-friendly fuels i.e., hydrogen and biogas.
Conclusions
Crude glycerol is the major by-product of biodiesel production process equivalent to
10% by weight. Due to global warming concerns and strict policies of leading nations,
global biodiesel production is rapidly increasing. However, such increase demands a
204
sustainable method for management of glycerol rich biodiesel manufacturing waste.
Alternatively, crude glycerol from biodiesel production plant can be used as a feedstock
for biological hydrogen production.
In the present investigation, techno-economic
feasibility of a two-stage fermentation process for CG bioconversion and hydrogen
production has been evaluated. Considering the materials, power, facility and labour
requirement, total cost associated with the process was calculated. From this analysis, it
was observed that the media components required for the process covered nearly 82 %
of total production cost. Based on the analysis, few propositions have been developed
for reduction of the process cost. Further, the process was found to be very beneficial
from environmental point of view. It was observed that bioconversion of 1 Kg of CG has
the potential to reduce nearly 7.66 Kg of GHG. Moreover, the process has the potential
to mitigate possible environmental hazards due to improper disposal of CG.
Abbreviations
CFR (Code of federal regulations), CG (crude glycerol), CSTR (continuous stirred tank
reactor), EPA (Environmental protection agency, US), GHG (greenhouse gas), kWh
(kilowatt hour), MSDS (material safety data sheet), RFS (Revised renewable fuel
standard)
Acknowledgments
The authors are sincerely thankful to the Natural Sciences and Engineering Research
Council of Canada (Discovery Grant 355254), Le Centre de recherche industrielle du
Québec (CRIQ), MAPAQ (No. 809051) and Ministère des Relations internationales du
Québec (coopération Paraná-Québec 2010-2012) for financial support. The views or
opinions expressed in this article are those of the authors.
205
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from
glycerol-containing
wastes
discharged
after
biodiesel
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glycerol
derived
from
biodiesel
manufacturing.
Siswo S (2010) Biogas production using anaerobic biodigester from cassava starch
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Table 2.4. 1: Feedstock availability and annual biodiesel and CG production in North America
Item
Annual production (MMt / million liter)
US
Canada
Total
Soya bean oil
8.6 MMt
0.24 MMt
8.84 MMt
Canola oil
0.46 MMt
1.6 MMt
2.06 MMt
Animal fat (Tallow)
2.53 MMt
0.2 MMt
2.73 MMt
Biodiesel
2839.05 million liter
190 million liter
3029.05 million liter
CG
0.181 MMt
0.019 MMt
0.2 MMt
210
Table 2.4. 2: Cost distribution of crude glycerol (1 Kg) bioconversion and hydrogen production using a two-stage process
SL No.
Process
Item required
Amount
Cost $
1.
Inoculum development (20 L)
Glucose monohydrate (5 g/L)
100 g
4.6
Casein peptone (5 g/L)
100 g
22.51
Yeast extract (0.5 g/L),
10g
1.2
KH2PO4 (2 g/L),
40g
8.15
MgSO4.7H2O (0.5 g/L)
10g
0.45
L-cysteine hydrochloride (0.5 g/L)
10g
7.97
Sterilization & incubation (17 h)
Electricity 0.85 kWh
0.037
2.
Pretreatment of CG
HCl
200 ml
2.38
3.
Media preparation (380 L)
Na2HPO4 (4.58 g/L)
1740.4 g
96.97
NaH2 PO4.H2O (2.45 g /L)
931 g
38.82
Yeast extract (1g/L)
380 g
25.25
4.
Anaerobic dark fermentation
Sterilization & incubation (48h)
Electricity 48 kWh
2.14
5.
Photo-fermentation
Inoculum development (20 L)
Same to dark fermentation
44.92
211
SL No.
Process
Item required
Amount
Cost $
NaOH
200 g
1.46
Yeast extract (0.5 g/L)
190 g
24.42
Illumination and incubation (144 hours)
Electricity 489.6 kWh
21.83
6.
Treatment of waste
Biogas will be produced
4010.9 L
(-) 3.27
7.
Equipment and labour
10 % additional cost
-
29.98
212
Table 2.4. 3: Hydrogen production by different fermentation techniques using crude glycerol as a cheap substrate
Fermentation
Inoculum
Experiment conditions
Initial
CG
concentration
Incubation
Incubation
time (h)
temperature
(g/L)
Dark fermentation
Thermotoga
(pure culture)
DSM 4359
Enterobacter
neapolitana
aerogenes
05
References
1.98 ± 0.10 mol-H2 mol-1
(Ngo
glycerol
2011)
(ºC)
200
75
et
al.,
10
12
37
63 mmol-H2 L-1 h-1
(Ito et al., 2005)
20
-
37
2448.27 ml-H2 L-1 medium
(Marques et al.,
HU-101
2009)
Dark fermentation
Anaerobic sludge
COD 3.67
25
25.0±0.5
200 ml-H2 g-1 COD
(Fernandes
et
al., 2010)
(mixed culture)
Heat treated mixed culture
03
72
30
(P. A. Selembo
et al., 2009a)
Photo
fermentation
(pure culture)
Rhodopseudomonas
~ 0.9
240
30
palustris
(SabourinProvost
et
al.,
et
al.,
2009)
Microbial electrolysis
Enterobacter
cell (pure culture)
NBRC 12010
aerogenes
09.9
36
30
(Sakai
2007)
213
Fermentation
Inoculum
Experiment conditions
Initial
CG
concentration
Incubation
Incubation
time (h)
temperature
(g/L)
Microbial electrolysis
Domestic wastewater
01
References
(Priscilla
(ºC)
36
30
A.
Selembo et al.,
cell (mixed culture)
2009b)
214
Table 2.4. 4: Estimation of environmental benefit from bioconversion of 1 Kg crude glycerol using a two-stage process
GHG emission sources
GHG emission
References
GHG emission from electricity consumed during bioconversion of
1.07 Kg of CO2eq
Present
1 Kg CG
estimation
and
(http://www.ec.gc.ca/ges-
ghg/default.asp?lang=En&n=EAF0E96A-1.
Aaccessed
27
Dec 2011)
GHG emission due to permanent constructions (10 % of CG
0.107 Kg CO2eq
bioconversion process)
GHG emission potential of 1 Kg CG (if disposed or used for other
0.65 Kg CO2eq
purpose)
Present estimation and (Carbon Life Cycle Calculation for
Biodiesel, Home Grown Cereals Authority, United Kingdom.
http://www.co2star.eu/final-documents/Annex3-4-WP2D3_Life%20cycle%20biodiesel.pdf. Aaccessed 27 Dec 2011)
GHG emission during combustion of hydrogen and biogas
Negligible
Present estimation
0.77 Kg CO2 eq
Present estimation and (Sheehan et al., 1998)
obtained from 1 Kg CG
GHG emission during production of 2.56 L (equivalent to 1 Kg
CG) fossil diesel
215
GHG reduction by replacing fossil fuel combustion with H2 and
7.42 Kg CO2eq
biogas from 1 Kg CG
Present
estimation
and
(http://hypertextbook.com/facts/2005/MichelleFung.shtml.
Aaccessed
27
Dec
2011;
http://hypertextbook.com/facts/2006/TatyanaNektalova.shtml.
Aaccessed
27
Dec
2011;
http://www.abc.net.au/tv/carboncops/factsheets/cc_cars.pdf.
Aaccessed 27 Dec 2011)
Total GHG reduction by 1 Kg CG bioconversion process
7.66 Kg CO2eq
216
Present estimation
Table 2.4. 5: Table summarizing possible benefits of the crude glycerol bioconversion process considered in the present study
Crude glycerol
Estimated H2
Estimated biogas
Estimated fossil fuel
Estimated GHG
Estimated value of
production
production
replacement
reduction (Kg
the process
CO2eq)
1 Kg CG
45.6 g
4010.9 L
2.56 L
7.66 Kg
$ 3.49
CG produced in
09.12 million Kg
802180 million liter
512 million liter of diesel
1532 million Kg
$ 697.34 M/year
NA/year
217
Figure 2.4. 1: Schematic representation of the process considered for the present estimation
Figure 2.4. 2: Cost distribution of the crude glycerol bioconversion process
219
Figure 2.4. 3: Electricity requirement of a two-stage fermentation process for CG bioconversion
and H2 production
220
CHAPTER 3
HYDROGEN PRODUCTION BY CRUDE GLYCEROL BIOCONVERSION:
A PRACTICAL SUMMARY
Chapter 3. Hydrogen production by crude glycerol bioconversion: a practical summary
PART I
HYDROGEN PRODUCTION FROM MEAT PROCESSING AND
RESTAURANT WASTE DERIVED CRUDE GLYCEROL BY ANAEROBIC
FERMENTATION AND UTILIZATION OF THE SPENT BROTH
Saurabh Jyoti Sarmaa, Satinder Kaur Brara, Yann Le Bihanb, Gerardo Buelnab,
Carlos Ricardo Soccolc
a
INRS-ETE, Université du Québec, 490, Rue de la Couronne, Québec(QC), Canada
G1K 9A9 bCentre de recherche industrielle du Québec (CRIQ), Québec(QC), Canada
c
[email protected])
Journal of Chemical Technology and Biotechnology, 88 (12): 2264–2271
223
Résumé
CONTEXTE: Le glycérol brut (GB), le principal sous-produit de processus de production
de biodiesel pourrait être utilisé pour la production de biohydrogène. Cependant, la
production d'hydrogène par fermentation est fortement limitée par le coût des solutions
tampons et des nutriments supplémentaires nécessaires à la fermentation. Ainsi, le but
de la présente étude était de déterminer le potentiel maximal de production de H2 à
partir du GB et ce, en l'absence de tout supplément coûteux supplémentaire. De plus, la
valorisation des rejets de fermentation (post production de H2) est un autre objectif qui a
été abordé.
RÉSULTATS: Un maximum de production de H2 de 2022,5 mL/L milieu de culture a été
réalisé par bioconversion du GB seul (sans éléments nutritifs supplémentaires). Une
concentration de 10 g/L de GB a été jugée optimale. En outre, l'addition d’une petite
quantité de 50 mg/L de biomasse microbienne en phase endogène (carencé) provenant
d’une fermentation antérieure a permis d’améliorer la production de 32,50%, avec un
taux maximum de 1,040 mL/L/jour. De même, près de 75% du H2 totale a été produit à
un pH aussi bas que 3,8, indiquant une tolérance élevée au milieu acide par la souche
Enterobacter aerogenes NRRL B407 utilisée.
CONCLUSION: Le glycérol brut provenant des procédés de transformation des
graisses animales et des déchets de restaurant a été caractérisé et évalué pour son
potentiel de production de H2. L'utilisation d’une biomasse âgée en carence de substrat
provenant d’une fermentation antérieure (comme complément) a permis d’améliorer les
performances du procédé.
Mots clés: biodiesel, glycérol brut, production d'hydrogène, pH, culture carencée
224
Abstract
BACKGROUND: Crude glycerol (CG), the major by-product of biodiesel production
process could be used for biohydrogen production. However, fermentative hydrogen
production is highly limited by the cost of buffer and additional nutrients required for the
process. Thus, the purpose of the present study was to determine maximum H 2
production potential of CG in the absence of any additional expensive supplement.
Further, sustainable utilization of the waste of H2 production process was the other
objective.
RESULTS: A maximum production of 2022.5 mL H2/L media has been achieved by CG
bioconversion (without any additional nutrient) and 10 g/L CG was found to be optimum.
Further, addition of spent biomass (50 mg/L) of the process into a subsequent process
was found to improve the production by 32.50% with a maximum rate of 1040 mL/L/d.
Similarly, nearly 75% of total H2 were produced at a pH as low as 3.8, indicating high
acid tolerance of the strain (Enterobacter aerogenes NRRL B407) used.
CONCLUSION: Meat processing and restaurant waste based CG has been
characterized and evaluated for maximum H2 production potential. Likewise, utilization
of spent biomass of CG bioconversion process (as supplement) was found to improve
the process performance.
Key words: Biodiesel, crude glycerol, hydrogen production, pH, spent broth
225
Introduction
Owing to the concern of global warming and limited storage of fossil fuel, global
production of biodiesel is rapidly increasing (Hasheminejad et al., 2011, Pachauri et al.,
2006). European Union and USA are the two leading biodiesel producers and presently
they have 120 and 176 active biodiesel manufacturing facilities, respectively (Sarma et
al., 2012). Further, biodiesel production has been promoted by different governments
through various subsidies and policies. US government has devised the revised
renewable fuel standard (RFS2) program, according to which by 2022, approximately 36
billion gallons of renewable fuel has to be blended into U.S. diesel fuel (Sarma et al.,
2013). Similarly, Canadian government has set a target of blending the diesel with at
least 2 % renewable fuel and 17 % reduction of greenhouse gas emission by 2020
(Sarma et al., 2013). Therefore, further increase in global biodiesel production is
expected in the coming years. Biodiesel is mainly produced by the transesterification of
vegetable oil and animal fats (Endalew et al., 2011). However, increased application of
these materials for biodiesel production may increase the price of these materials in
food market (Mata et al.). Therefore, low cost materials such as, the waste of meat
processing industry containing animal fat and waste cooking oil from restaurants are
becoming popular as the substrate for biodiesel production (Mata et al., Meng et al.,
2008). During biodiesel production, for every 100 Kg of biodiesel, 10 Kg of crude
glycerol (CG) is generated as by-product (Selembo et al., 2009, Yazdani et al., 2007).
However, due to the presence of large amount of impurities, unlike its pure counterpart,
CG has very low commercial value (Pachauri et al., 2006). Moreover, CG derived from
complex waste materials such as, meat processing and restaurant waste may have
even more impurities than the CG derived from pure substrates and its commercial
application will be further limited. Further, direct release of CG to the environment,
without proper treatment, is known to be unacceptable for the environment (da Silva et
al., 2009, Selembo et al., 2009). Alternatively, CG is a good carbon source and its
application for fermentative hydrogen production may be a sustainable means for its
value added utilization (Ngo et al., 2011, Selembo et al., 2009). Likewise, water is the
226
only major byproduct of H2 combustion process (Selembo et al., 2009) and hence, the
proposed process will contribute in reducing the greenhouse gas emission.
As already mentioned, due to high glycerol content, CG is mainly a carbon source and
hence, addition of other nutrient source is required for its enhanced bioconversion.
Further, during H2 production a range of organic acids are produced to change the
media pH (Jame et al., 2011) and the process quickly approaches towards termination.
Therefore, the media is supplemented with buffering agents to avoid any drastic pH
change. However, we have made a techno-economic evaluation of the process (Sarma
et al., 2013) and found that the cost of the buffer and synthetic media components used
for the process may be as high as 82% of total process cost. Similarly, during H2
production by bioconversion of 1Kg of CG, 400 L of spent media will be generated
(Sarma et al., 2013) which will be composed of a range of organic acids, residual CG,
residual nutrient and biomass. Thus, the present investigation has three objectives- (i)
evaluation of H2 production by bioconversion of meat processing and restaurant waste
derived CG without applying any buffering agent and synthetic media components, (ii)
screening of less expensive nutrient source for the process and; (iii) utilization of spent
broth of the process. Further, Enterobactor aerogenes is a well-known microorganism
for its CG bioconversion and H2 production ability (Ito et al., 2005, Marques et al., 2009,
Sakai et al., 2007) and it was considered for the present investigation. Thus, it is an
integrated study to improve the CG bioconversion process in terms of H 2 yield, as well
as to develop a sustainable process for utilization of the spent media generated during
H2 production to develop a minimum waste sustainable process which can be integrated
into the glycerol refinery in a typical biodiesel production plant.
Materials and methods
Microorganism and inoculum development
The microorganism considered for this study is Enterobacter aerogenes NRRL B-407,
collected from ARS, USDA, USA. It is a gram-negative, rod-shaped facultative
anaerobic bacteria belonging to the family Enterobacteriaceae. A complex media
containing glucose (5 g/L), casein peptone (5 g/L), KH2PO4 (2 g/L), yeast extract (0.5
227
g/L) and MgSO4.7H2O (0.5 g/L) was used to grow and maintain the microorganism
(Beckers et al., 2010). For this purpose, 125 mL serum bottles (working volume 50 mL)
were used and anaerobic environment was created by sparging the media with pure N2
gas for 2 min. The culture was incubated in orbital incubator shaker maintained at 150
rpm and 30°C (Marques et al., 2009) and the log phase culture broth was used as
inoculum throughout the study.
Chemicals and reagents
Crude glycerol (CG) used in the present study was collected from Rothsay®, Canada.
Rothsay uses the fat containing waste from meat processing plants and used grease
from restaurants to produce biodiesel (http://www.rothsay.ca/products/biodiesel/,
accessed on 19/07/2013). Chemicals, such as glucose, MgSO4.7H20, NaOH, sodium
periodate, acetylacetone, ethanol and acetic acid were purchased from Fisher scientific,
Canada. Casein peptone and KH2PO4 was purchased from VWR, Canada. Yeast
extract used in the present investigation was a gift from Lallemand, Canada.
Characterization of CG
Glycerol content and physical properties
Glycerol content of the CG was determined by UV-Vis spectrophotometer and the
detailed method has been separately described later. The density of crude glycerol was
determined from the volume and weight of crude glycerol measured at room
temperature (24 ± 0.5 °C) (Hu et al., 2012). Similarly, to determine the pH, 1 g of crude
glycerol was dissolved in 50 mL of deionized water and at room temperature (24 ± 0.5
°C), the pH of the solution was measured by a digital pH meter (EcoMet®, Istek Inc.) (Hu
et al., 2012).
Elemental analysis
The CG was analyzed for different elements, such as Na, K, Fe, Ca, Mg, Mn, Al, P, Co,
Cu, and Zn by using ICP-AES (Varian VISTA-AX, CCD simultaneous ICP-AES). Prior to
the analysis, the sample was digested by a method slightly modified from the original
method
used
for
acid
digestion
228
of
soil
and
sediment
(http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/3050b.pdf). Briefly, 500 mg of
sample was taken in a glass tube and placed in a block digester (Technicon BD-40).
About 5 mL of HNO3 (50%) was added to the sample, the temperature of the block was
increased up to 95 ± 1°C and left for 10 min. After removing the tube, it was allowed to
cool down for 15 min and 2.5 mL of concentrated HNO3 was added. Later, the tube was
again incubated for 1 hour at 95 ± 1°C, which was subsequently allowed to cool down
for 30 min. Further, 3 mL H2O2 was added and the tube was incubated for 10 min at the
same condition as already mentioned and allowed to cool down for 5 min. This step was
repeated two more times following which the tube was left for two hours at 95 ± 1°C.
After this period, 2.5 mL of concentrated HNO3 was added and the tube was again left
for 15 min in the same digestion block operated at 95 ± 1°C. Finally, the sample was
cooled down and the volume was adjusted to 50 mL using distilled water before
analysis.
Carbon and nitrogen content of the CG sample was analyzed by total organic
carbon/total nitrogen measuring unit (Shimadzu TOC-VCPH/TNM-1) using standard
protocol
(http://www2.shimadzu.com/apps/appnotes/tocan102.pdf).
The
important
conditions of the analysis were as follows: injection volume (500µL), sparging time (7
min), carrier gas flow rate (150 mL/min) and furnace temperature (720 °C).
Investigation of the effect of CG concentration on hydrogen production
To determine the effect of initial CG concentration on hydrogen production, five different
concentrations of CG were tested, viz., 2.5 g/L, 5g/L, 10 g/L, 15 g/L and 20 g/L. Similar
to inoculum development, 125 mL serum bottles (working volume 50 mL) were used for
hydrogen production experiments and CG was diluted with deionized water to achieve
different initial concentrations. At pH 6.0, high hydrogen production by the
microorganism E. aerogenes has been reported (Sakai et al., 2007) and hence, in all
cases, the pH of the media was adjusted to 6.0. Media was sparged with pure N2 gas as
already described and the bottles were sealed with aluminum crimp seal containing
silicone septum (Fisher scientific, Canada). After autoclaving, the media was allowed to
cool to room temperature and inoculated with 5 % (v/v) inoculum equivalent to 0.25 mg
229
dry cell mass. Bottles were incubated in the same way as described in previous section
and 1 mL of aqueous sample was collected at 12 h interval. Composition of the
produced gas was analyzed by gas chromatography; the volume of produced H2 was
measured at 12 h interval as described later.
Characterization of spent media fraction
To determine the cell mass present, 10 mL of the spent media was centrifuged at 5000
x g for 15 min, cell pellet was collected, dried at 105° C, weighed and the value was
expressed as mg dry cell/L media (Chang et al., 2005). pH of the broth was determined
by a digital pH meter (EcoMet®, Istek Inc. Korea). The spent broth was also subjected to
an elemental analysis including carbon and nitrogen content using ICP-AES and
TOC/TN analyzer, respectively. The method of sample preparation and analysis used
for this investigation was similar to that used for CG and already discussed in details.
Utilization of spent media fraction in subsequent process for H2 production
To evaluate the possibility of utilizing the spent media of H2 production process for CG
bioconversion, a separate set of experiment was carried out. Media containing 10 g/L
CG (pH: 6.0) was prepared, inoculated with E. aerogenes and incubated for 7 d as
stated previously. The spent broth generated from this batch process was mixed with 10
g/L sterile CG taken in different serum bottles, at different proportions (2, 5, 10 and 20
% v/v) however, the final working volumes of all the reactors were kept constant at 50
mL. All the bottles were inoculated with 5% (v/v) inoculum, however, a control
experiment was also carried where, 10 g/L CG was mixed with only spent broth (5 %
(v/v) and no fresh inoculum was used. As during first cycle of fermentation, CG was
almost completely utilized, the final glycerol concentration in all the culture bottles was
nearly constant. Further, the incubation conditions and sampling interval were same as
already described.
Utilization of active biomass from spent media
To evaluate the possibility of recycling the living biomass of H2 production process, a
small scale batch H2 production experiment was carried out. Five different serum bottles
230
containing 10 g/L CG were prepared in the same way as already described. Further, the
spent broth of CG (10 g/L) fermentation was centrifuged at 4000 x g for 5 min at 4°C
(Sorvall® RC 5C plus, Sorvall instrument, Du pont) and the cell pellet was collected.
Collected biomass ranging from 5 to 30 mg/L (equivalent dry cell mass) was mixed with
5 % (v/v) fresh inoculum and distributed into all the different serum bottles containing
sterilized media. However, similar to the previous study, one control experiment was
performed using active biomass equivalent to 5 mg dry cell mass/L, where no fresh
inoculum was used. All the bottles were incubated and the gas and liquid samples were
collected and analyzed in the same way as described in previous sections.
Utilization of spent biomass as a supplementary nutrient
To evaluate the possibility of recycling the residual biomass of H2 production process,
E. aerogenes cells were grown in a media containing 10 g/L CG, using the same
experimental setup and conditions used for H2 production experiments. After 7 days of
fermentation, the cells were harvested by centrifugation (5000 x g for 5 min) and mixed
with 10 g/L CG at different concentrations ranging from 5 to 150 mg/L. The different
serum bottles containing the media and the cell pellet was then sterilized, inoculated
and incubated in the same way as described in previous sections.
Analysis
Glycerol analysis
During hydrogen production experiments, residual glycerol concentration was measured
by the spectrophotometric method described by (Bondioli et al., 2005). The method is
based on periodate oxidation of glycerol where, 1 mL of sample was mixed with 1 mL of
ethanol (47.5% v/v) followed by the reaction with 1.2 mL each of acetylacetone solution
(0.2 M) and sodium periodate solution (10 mM) at 70 ± 1°C for 1 min. Following the
reaction, the samples were immediately cooled to 20-25 °C using tap water and
maintained in the same temperature until analyzed using a Uv-Vis spectrophotometer
(Carry 100 Bio®, Varian USA) at 410 nm.
231
Hydrogen analysis
The composition of produced gas was analyzed by a gas chromatograph (Varian 3800,
USA) fitted with PoraPLOT Q® column (Agilent technology, USA) and thermal
conductivity detector (TCD). The column temperature was 100 °C and Nitrogen was
used as the carrier gas. The gas flow rate used in this analysis was 3.5 mL min -1 and
the retention time was 4.5 min. The volume of produced H2 was measured by the
inverted cylinder –NaOH (10%) method as described by previous researchers
(Junyapoon et al., 2011, Maeda et al., 2008). Briefly, the produced gas was passed
through 100 mL NaOH (10% w/v) and collected in a graduated glass cylinder filled with
water and placed in inverted position in a beaker containing water. Volume of H2
produced was calculated from the displacement of water level in the cylinder
(Junyapoon et al., 2011, Maeda et al., 2008) and, where applicable, considering the
temperature and atmospheric pressure, volume of produced gas was converted to
mmol (http://www.easycalculation.com/chemistry/ideal-gas.php).
Results and discussion
Crude glycerol characterization
The meat processing waste and waste grease derived CG fraction used in the present
study was found to be composed of at least 23.63 ± 2.5 % (w/w) glycerol and the
detailed characterization of the CG sample is presented in Table 3.1.2. The glycerol
content of the CG sample investigated in this study was found to be lower than used
food oil based CG (45 % glycerol, w/w) (Sakai et al., 2007) and soybean oil based CG
(63.3 % glycerol, w/w) (Hu et al., 2012). However, total organic carbon content (35.9 %
(w/w) ± 0.4) is notably higher than the total glycerol content of the CG investigated,
indicating the presence of considerable amount of carbonaceous material other than
glycerol. Similarly, compared to soybean oil based CG (0.3 ± 0.1 %, w/w), relatively
higher nitrogen content (3.25 ± 0.1 %, w/w) has been observed; however, the finding is
still comparable to soybean oil- waste vegetable oil based CG (1.2 ± 0.1%, w/w) (Hu et
al., 2012). The difference in nitrogen content indicates the possible presence of
proteinaceous materials in the meat processing and restaurant waste grease derived
232
CG. Further, from the elemental analysis, presence of relatively higher amount of S
(8827 ± 33 ppm) has been observed for the first time (as per the knowledge of the
authors) in a CG sample. Moreover, the Fe content of the CG sample was also higher
than soybean oil derived CG (Table 3.1.2). Coincidently, these two elements are the
core structural components of Fe-S clusters of major H2 producing enzymes
(hydrogenases) (Kim et al., 2011) and play a vital role in electron transfer during H2
production (Marseille, 1998). Their presence, therefore, might have beneficial effect for
a CG bioconversion process dedicated for H2 production. Similarly, pH of the CG
sample was found to be 3.4 (at 24 ± 0.5 °C), which is significantly lower than different
reported values for soybean oil based CG (pH 6.9), waste vegetable oil based CG (pH
10.1) as well as pure glycerol (pH 6.4) (Hu et al., 2012, Sakai et al., 2007). Interestingly,
such a low pH was not a problem for hydrogen production as CG was always diluted
with distilled water before bioconversion process and the resulting solution had a pH of
~5.5. Further, the density of CG was determined to be 1106 g/L which is nearly similar
to different reported values (Table 3.1.2) and lower than pure glycerol (Hu et al., 2012).
Hydrogen production by CG bioconversion
CG at different concentrations (2.5 to 20 g/L) was tested as a feedstock for H2
production and different profiles are presented in Figure 3.1.1. From Figure 3.1.1, it can
be seen that for all concentrations, the rate of H2 production was identical in the first 24
h. Further, in the case of 2.5 g/L CG, no H2 production was observed after 24 h of
incubation. Similarly, in the case of 5 g/L CG, the rate of H2 production decreased after
48 h and no production was observed after 60 h. For 10, 15 and 20 g/L CG, H2
production rates were almost similar and constant up to 60 h which were then
decreased and no significant production was observed after 168 h of incubation. From
these observations, it can be concluded that by increasing the CG concentration from
2.5 g/L to 10 g/L, the cumulative H2 production can be increased from 580 mL/L to
2022.5 mL/L media. However, when the concentration was increased from 10 to 20 g/L,
no significant improvement in H2 production was observed. Such observation may be
due to substrate inhibition or the impurities of CG, some of which are known to inhibit
microbial growth. Similarly, Figure 3.1.1 shows the profiles of glycerol utilization during
233
H2 production by using different concentrations of CG. From the Figure 3.1.1, it can be
observed that in the case of 2.5 g/L CG, within 36 h, the glycerol was almost completely
utilized by the microorganism. Similarly, in the case of 5 g/L CG, it was almost
completely utilized within 120 h and in case of 10 g/L, 94.08 % glycerol was consumed
within 7d. However, in the case of 15 and 20 g/L CG, only 71.96 and 59.05 % of the
glycerol was utilized during the investigation period (7d). Therefore, considering both
H2 production and glycerol utilization, 10 g/L CG is chosen to be the optimal
concentration to be used for further investigations.
Further, as the feedstock used in the present investigation is a waste material and
contains different impurities; hence, pretreatment of the substrate may be needed prior
to its application as a fermentation media. Methanol and solid particles present in the
CG can be removed by rotary evaporation and centrifugation, respectively (Ngo et al.,
2011). Similarly, soap present in CG may be inhibitory for microbial growth and can be
removed by precipitation using acid followed by centrifugation (Athalye et al., 2009, Chi
et al., 2007). Similarly, as shown in Table 3.1.1, fermentation media can be
supplemented with different synthetic media components. However, these additional
steps of pretreatment and addition of supplementary nutrients can further increase the
process cost. Hence, another objective of the present investigation was to evaluate H2
production without any substrate pretreatment and synthetic media component.
Moreover, the maximum cumulative H2 production obtained in this investigation has
been compared with different literature reports and presented in Table 3.1.1.
Productivity of the present system was found comparable to different recent
investigations and more importantly, addition of a large amount of supplementary media
components and additional feedstock preparation have been successfully avoided
without compromising the product yield (Table 3.1.1).
Figure 3.1.2 represents the pH profile of the media during H2 production using different
concentrations of CG. From the profiles, it is observed that in all cases, within first 24 h,
the media pH dropped to below 4. Further, there is a correlation between the media pH
at the end of the process and the initial CG concentrations. In the case of 20 g/L CG,
the final pH of the broth was as low as 3.65; however, in the case of 2.5 g/L CG, the pH
234
was 4.06. Such observation may be due to the fact that in the case of 20 g/L CG, the
substrate was available to the microorganism till the end of the fermentation and the
amount of different organic acids produced was more than that of 2.5 g/L CG. In the
latter case, the substrate was completely utilized within 24 h and the amount of organic
acid produced was not sufficient to drop the pH below a certain level. Further, in this
case, towards the end of the incubation period, the media pH was increased from 3.74
to 4.06. A possible reason for such observation may be that in the absence of available
carbon source, the organic acids produced during first 24 h were utilized by the
microorganism to increase the media pH. However, it is only an assumption and further
investigation will be necessary to prove it. Further, as shown in Table 3.1.1, different
buffering agents are commonly added to the CG based culture media used for H2
production (Ngo et al., 2011, Selembo et al., 2009). The main reason of such practice is
to prevent any drastic change in media pH due to simultaneous production of different
organic acids during H2 production, in turn, which may inhibit the process. Figure 3.1.3
represents the H2 production and pH profiles for 10 g/L CG. It can be seen that nearly
75 % of total H2 was produced when the media pH was around 3.8. Interestingly,
cumulative H2 production is still comparable with different recent investigations carried
out in the presence of buffering agents (Table 3.1.1). Therefore, it can be concluded
that E. aerogenes can continue H2 production in a comparable rate at a pH as low as
3.8 and addition of buffering agents to the process may be avoided. This observation
may be important for future commercialization of the process, where the requirement of
a large amount of buffering agents could be avoided to boost the economic feasibility of
such processes.
Further, to have an idea about the importance of the waste materials generated during
the process, a mass balance analysis has also been made. From the analysis, it was
observed that during hydrogen production process, 10.65 g of media components (CG
and the constituents of inoculum) present in 1L of media is converted to 50 mg dry cell
mass and about 169.4 mg H2 gas. Rest of the material is either unutilized nutrients or
different organic acids, solvents and waste gases (mostly CO2) generated during the
235
process. Thus, this observation indicates that recycling of the waste liquid and gas is
necessary for maximum utilization of the CG.
Utilization of spent broth of H2 production process
Unlike most industrial processes where the fermented broth is subjected to downstream
processing for recovery of the final product, during H2 production, the gas is readily
separated from the media and the fermented broth has no further use. Moreover, as
already mentioned, 400 L of such spent media (SM) may be generated for every Kg of
CG used (Sarma et al., 2013). Further, the SM of the process may contain the living and
dead biomass, residual nutrients including glycerol and biomolecules such as, proteins
released to the media during fermentation and a detail characterization of the spent
media is presented in Table 3.1.2. Further, it may be possible that the living biomass of
SM still retains its H2 production potential and the residual nutrients and biomolecules
present in the broth can be used as a supplementary nutrient source for a subsequent
H2 production process. Unfortunately, because of the acidic pH and negligible glycerol
content (Table 3.1.2), such spent broth will not be suitable as a sole medium component
for a subsequent process. However, if the SM is mixed in small quantity with a fresh
media containing CG, improved H2 production may be observed for this new process.
Based on this assumption, as already mentioned, an experiment was carried out where
10 g/L CG was mixed with different volumes of spent broth and the result of this
investigation is shown in Figure 3.1.4. Figure 3.1.4 shows H2 production profiles for the
different runs of the experiment. From Figure 3.1.4, it can be seen that SM fraction (5%
v/v), when used without any fresh inoculum can also produce H2 by using a media
containing only CG (10 g/L). However, the level of H2 production was very low.
Interestingly, when fresh inoculum was used along with the SM, the production was
increased by several folds. Similar results were observed for different SM fractions
ranging from 2 to 10 % v/v, however, when the SM fraction was increased to 20 % (v/v),
production was inhibited. Similar results were observed when the SM fraction was
increased beyond 20 % (data not shown).
236
Further, as compared to H2 production by using only CG (10 g/L) (Figure 3.1.1), addition
of SM (2-10 % v/v) was found to enhance the production by 13.37 %. As already
discussed, there may be two reasons for such observation; firstly, in addition to the
living cells present in inoculum, the SM also had living E. aerogenes cells which might
help in enhanced H2 production. Similarly, CG is mainly a carbon source, hence,
proteins and other nutrients present in SM might help in increased cell proliferation
leading to enhanced H2 yield.
Further, as elaborated in Table 3.1.1 and already
discussed, cumulative H2 production obtained in the present study is comparable with
different investigations carried out in the presence of expensive buffering agents and
synthetic supplementary nutrients.
From the previous study, we found that if appended with fresh media, the active
biomass (AB) present in the spent media of H2 production process is still has the
potential to produce H2. However, if the spent media containing the living cells are
directly mixed with fresh media in small quantity, the number of viable cells present in
this volume may not be sufficient for significant improvement in H2 production.
Moreover, when larger volume of such spent media was used (20 % v/v or beyond), it
was found to be inhibitory for H2 production. Therefore, it was decided that the cells will
be separated from the spent by centrifugation and such cell pellet will be added to the
fresh media, instead of whole spent broth. For this purpose, AB ranging from 5-30 mg/L
was tested and the results are presented in Figure 3.1.5. From Figure 3.1.5, it can be
seen that the cell pellet (5 mg/L) collected form spent broth, when mixed with fresh
media was also able to produce a small amount of H2. However, upon mixing the cell
pellets with fresh inoculum (5% v/v), H2 production was greatly improved. Further, there
is a correlation between the amount of cell pellet used and the cumulative H2
production. When the amount of cell pellet used was increased from 5 mg/L to 30 mg/L,
production of H2 was found to be improved by 57.98% (Figure 3.1.5).
Furthermore, as a fresh media containing 10 g/L CG was used for all these
investigations, the H2 production profile obtained for 10 g/L CG (Figure 3.1.1) was
considered as the control profile and the results were compared. From this comparison,
it was observed that, in the case of 5 mg/L and 10 mg/L AB, the amount of H2 produced
237
was lower than the control. The possible reason for this observation may be that prior to
the inoculation, the inoculum used in this investigation was mixed with the cell pellet
from previous fermentation and this step might alter the efficiency of the inocula.
However, when the addition of cell pellet was increased to 30 mg/L, due to higher cell
density in the inoculum, improved H2 production was observed over the control profile.
Microbial cells are good source of protein (Dhanasekaran et al., 2011) and it may be
possible to use them as a nitrogen source during fermentation. As already mentioned,
CG is mainly a carbon source and addition of a nitrogen source may result in improved
H2 production. Therefore, one more investigation was carried out to evaluate the
possibility of utilizing the SB of H2 production process as a nitrogen/nutrient source for
subsequent fermentation. Cell pellets of previous fermentation (5- 150 mg/L) was mixed
with the media containing 10 g/L CG and H2 production experiments were carried out as
described in materials and methods section. Figure 3.1.6 shows the H2 production
profiles obtained from this investigation. The results were compared with the profile
obtained for 10 g/L CG (without any supplementary nutrient, (Figure 3.1.1), and in the
case of 50 mg/L SB, cumulative H2 production was found to increase by 32.50%.
However, SB in the range of 5-10 mg/L was not sufficient to produce any significant
improvement in cumulative H2 production. Further, in the case of 150 mg/L SB, H2
production was improved by 25.59 %; however, the improvement was lower than that of
50 mg/L SB. The most probable reason for such observation may be that the addition of
150 mg/L SB might decrease the C-N ratio (increase the nitrogen content) of the media
to inhibit the process (Androga et al., 2011, Zhu et al., 1999). The results are compared
with different reports on H2 production by CG bioconversion and presented in Table
3.1.1. From Table 3.1.1, it can be concluded that SB (50 mg/L) of previous fermentation
can be reused as a supplementary nutrient for improved H2 production and a large
amount of synthetic media components can be replaced.
Conclusions
Significant difference in glycerol content, pH and elemental composition was observed
between soybean oil based and meat processing and restaurant waste based CG. 10
238
g/L of the latter was found to be optimum for H2 production (as feedstock). In this case,
nearly 75 % of the total H2 was produced when media pH was around 3.8, indicating the
possibility of H2 production in the absence of any expensive buffering agents. Further,
application of spent biomass (50 mg/L) as nutrient source for a subsequent process can
improve H2 production by 32.50% with a maximum rate of 1040 mL/L/d.
Acknowledgments
The authors are thankful to S. Duval, D. P. Veetil and L. Rabeb for their assistance in
sample collection and analysis. Financial support of the Natural Sciences and
Engineering Research Council of Canada (Discovery Grant 355254), Le Centre de
recherche industrielle du Québec (CRIQ), MAPAQ (No. 809051) and Ministère des
Relations internationales du Québec (coopération Paraná-Québec 2010-2012), has also
been sincerely acknowledged.
239
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242
Table 3.1. 1: Summary of different recent investigations on CG bioconversion and H2 production
Sl. no
Microorganisms
CG
Additional media components/buffering agent
H2 production
Reference
2.0 g/L soybean flour as the N-source, 0.05 M HEPES
518.25
(Ngo
buffer
concentration
(g/L)
1
Thermotoga
5.0
neapolitana
2
Enterobacter
20
7.0g of K HPO , 5.5g of KH PO , 5g of tryptone, 5g of
2
aerogenes ATCC
4
2
4
et
al.,
mL/L
2011)
2500 mL/L
(Marques
et
±
25.9
al., 2009)
yeast extract , 1.0g of (NH ) SO , 0.25g of MgSO .7H O,
4 2
13048
4
4
2
0.021g of CaCl .2H O, 0.12g of Na MoO .2H 0, 0.002g of
2
2
2
4
2
nicotinic acid, 0.000172g of Na SeO and 0.00002g of
2
3
NiCl per liter medium
2
3
Mixed culture from
COD 3.6
Sodium bicarbonate (500mg), chloridric acid (10 mol),
200 mL/g COD
al., 2010)
CH4N2O (6 mg), NiSO4∙6H2O(0.15 mg), FeSO4∙7H2O (0.75
anaerobic reactors
(Fernandes et
mg), FeCl3∙6H2O (0.075 mg), CoCl2∙2H2O (0.012 mg),
used for hydrogen
CaCl2∙6H2O (0.618 mg), SeO2
production
(0.0108 mg), KH2PO4
(1.608 mg), KHPO4 (0.39 mg), Na2HPO4∙2H2O (0.828 mg)
per liter
4
Mixed culture from
3.0
wheat soil
g/L)
(COD
1.3
70mM MES buffer, NH4Cl (0.31 g/L),
KCl (0.13 g/L), trace vitamins and minerals
243
43 mL/g COD
(Selembo
al., 2009)
et
Sl. no
Microorganisms
CG
Additional media components/buffering agent
H2 production
No additional media component
2022.5
Reference
concentration
(g/L)
5
mL/L
(equivalent
E. aerogenes
6
NRRL B407
to
84.08 mmol/L )
10
Present study
Spent biomass (50 mg/L)
2680
mL/L
(equivalent
111.42 mmol/L)
244
to
Table 3.1. 2: Characterization of crude glycerol and spent broth of H2 production process
Composition/property
Soybean oil based CG
Meat
processing
and
restaurant
Spent broth of CG (10
(from literature)
(grease) waste based CG (present
g/L)
study)
production
based
References
H2
(present
study)
Glycerol content
69.5 % / 63.0 ± 0.3 % (w/w)
23.63 ± 2.5 % (w/w)
0.014 % (w/w)
(Hu et al., 2012,
Selembo
et
al.,
2009)
pH
6.9
3.4 (24 ± 0.5 °C)
3.8 (24 ± 0.5 °C)
(Hu et al., 2012)
Density (g/L)
1010-1200
1106
-
(Hu et al., 2012)
Total carbon content %
24.3 ± 0.2
35.9 ± 0.3
2.88 ± 0.2
(Hu et al., 2012)
0.3 ± 0.1
3.25 ± 0.1
0.031 ± 0.1
(Hu et al., 2012)
S (ppm)
-
8827 ± 33
152.5 ± 28
-
Na (ppm)
11769 ± 1561
7508 ± 36
686.9 ± 35
(Hu et al., 2012)
P (ppm)
38.7 ± 4.8
1179 ± 20
837.9 ± 20
(Hu et al., 2012)
Ca (ppm)
Below detection limit
215.1 ± 19
191.6 ± 22
(Hu et al., 2012)
(w/w)
Total nitrogen content %
(w/w)
245
Composition/property
Soybean oil based CG
Meat
processing
and
restaurant
Spent broth of CG (10
(from literature)
(grease) waste based CG (present
g/L)
study)
production
based
References
H2
(present
study)
K (ppm)
118.8 ±26.8
125 ± 19
39.41 ± 18
(Hu et al., 2012)
Mg (ppm)
Below detection limit
57.41 ± 18
51.73 ± 18
(Hu et al., 2012)
Fe (ppm)
31.6 ±19.0
44.41 ± 16
5.13 ± 16
(Hu et al., 2012)
Zn (ppm)
-
~1.74
~1.05
-
Ni (ppm)
-
~0.94
~0.49
-
246
CG 2.5 g/L
CG 5 g/L
CG 10 g/L
CG 15 g/L
CG 20 g/L
CG 2.5 g/L
CG 5 g/L
CG 10 g/L
CG 15 g/L
CG 20 g/L
5
120
4.5
H2 production (mmol)
3.5
80
3
2.5
60
2
40
1.5
1
Residual glycerol conc. (%)
100
4
20
0.5
0
0
0
24
48
72
96
120
144
168
Time (h)
Figure 3.1. 1: Batch H2 production (solid lines) and glycerol utilization (dotted lines) profiles
obtained for different initial CG concentrations
4.8
CG 2.5 g/l
CG 5 g/l
4.6
Culture media pH
CG 10 g/l
4.4
CG 15 g/l
CG 20 g/l
4.2
4
3.8
3.6
0
24
48
72
96
120
Time (h)
144
168
192
Figure 3.1. 2: pH profiles obtained from the batch experiments conducted for different (initial) CG
concentrations
248
Hydrogen
pH
5
4.8
4.6
4
3.5
4.4
3
2.5
4.2
2
4
1.5
1
Culture media pH
4.5
3.8
0.5
0
3.6
0
24
48
72
96
120
Time (h)
144
168
192
Figure 3.1. 3: H2 production and pH profile obtained for 10 g/L CG showing the production of
nearly 75% of H2 at pH 3.8
249
6
5
4
Spent media
2 % v/v
Spent media
5 % v/v
Spent media
10 % v/v
Spent media
20 % v/v
3
2
1
0
0
24
48
72
96
120
Time (h)
144
168
192
Figure 3.1. 4: Batch H2 production profiles obtained by addition of different spent media fractions
250
5
AB 5 mg/L
without
inoculam
AB 5 mg/L
with inoculam
4.5
4
3.5
3
2.5
AB 10 mg/L
with inoculam
2
1.5
1
AB 30 mg/L
with inoculam
0.5
0
0
24
48
72
96
120
144
168
192
Time (h)
Figure 3.1. 5: Batch H2 production profiles obtained by addition of different amount of active
biomass from previous fermentation
251
7
6
5
4
SB 5 mg/L
3
SB 10 mg/L
2
SB 50 mg/L
1
SB 150 mg/L
0
0
24
48
72
96
120
144
168
192
Time (h)
Figure 3.1. 6: Batch H2 production profiles obtained by addition of different concentrations of
spent biomass (autoclaved) from previous fermentation
252
PART II
EVALUATION OF DIFFERENT SUPPLEMENTARY NUTRIENTS FOR
ENHANCED BIOHYDROGEN PRODUCTION BY ENTEROBACTER
AEROGENES NRRL B 407 USING WASTE DERIVED CRUDE
GLYCEROL
Louhichi Rabebc, Carlos Ricardo Soccold, Mhamdi Naceurc, Bouraoui Rachidc
a
b
c
Ecole Supérieure d'Agriculture de Mateur, Route de Tabarka - 7030 Mateur, Tunisia
d
[email protected])
International Journal of Hydrogen Energy 38 (5) 2013: 2191–2198
253
Résumé
Le glycérol brut (GB) provenant de l’industrie du biodiesel (graisse animal et de
restaurant) a plusieurs avantages comme substrat de culture pour la production de
biohydrogène par Enterobacter aerogenes NRRL B 407. Également, les coûts élevés
du processus de production en raison des additifs chimiques (nutriments) nécessaires à
la fermentation est une préoccupation. Par conséquent, des nutriments moins coûteux,
principalement composés de divers résidus d’origine agroalimentaire, ont été évalués
comme additifs supplémentaires pour augmenter la production de H2. Parmi les résidus
choisis, les rejets liquides d'abattoir (AL), la biomasse des déchets de brasserie (BDB)
et l'urée ont permis d’améliorer la production de 18,8 ± 3,6, 27,3 ± 3,5 et 38,6 ± 3,7%,
respectivement.
Dans le cas de l'urée (10 mg/L), une production cumulative aussi
élevée que 116,4 ± 3,7 mmol H2/L milieu a été obtenue, ce qui se compare à d'autres
recherches portant sur la bioconversion du GB. Ainsi, cette étude démontre que le
remplacement de nutriments chimiques conventionnels et coûteux, utilisés en grande
concentration (~ 5 à 6 g/L) par des rejets agroalimentaires ajoutés en quantité
négligeable (~ 10 mg/L), n’affecte pas les rendements cumulatifs en H2. Également, la
souche utilisée dans la présente étude a été apte à se développer à un pH acide aussi
bas que 3,3, indiquant de bonne perspective d’application pour la production de H2 par
fermentation sombre.
Mots clés: déchets de brasserie, glycérol brut, production d'hydrogène, éléments
nutritifs supplémentaires, urée
254
Abstract
Crude glycerol (CG) has several advantages over a range of conventional substrates
used for biohydrogen production by Enterobacter aerogenes NRRL B 407. Meanwhile,
high process cost due to requirement of expensive supplementary media component is
a concern.
Therefore, different less expensive (or wastes) materials have been
evaluated as supplementary nutrient for H2 production by CG (meat processing and
restaurant waste based biodiesel derived) bioconversion. Among the materials selected,
slaughterhouse liquid waste (SL), brewery waste biomass (BWB) and urea was found to
improve the production by 18.81 ± 3.56, 27.30 ± 3.54 and 38.57 ± 3.66 %, respectively.
Further, in the case of urea (10 mg/L), cumulative production as high as 116.41 ± 3.72
mmol H2/L media has been achieved; which is comparable to other reports available on
CG bioconversion. Thus, present study demonstrates successful replacement of large
amount (~5-6 g/L) of expensive nutrients/buffering agents by negligible amount (~10
mg/L) of different waste materials, without compromising the cumulative H2 yield.
Further, the strain used in the present study was found to grow at an acidic pH as low
as 3.3, indicating its prospective application for dark fermentative H2 production.
Key words: Brewery waste, crude glycerol, hydrogen production, supplementary
nutrient, urea.
255
Introduction
Hydrogen combustion does not produce CO2 (greenhouse gas) and water is the only
byproduct of the process. Therefore, it is projected as the cleanest future alternative of
the fossil fuels (Özgür et al., 2010). Steam reforming of natural gas and ethanol, coal
gasification and partial oxidation of heavy oil are a few methods available for hydrogen
production (Dicks, 1996, Vasudeva et al., 1996, Yurum, 1995). However, such
processes have different concerns, such as dependence on fossil fuels as well as
elevated thermal demand. Alternatively, bioconversion of organic feedstock is a
sustainable method for H2 production. A number of agricultural waste or plant based
materials, such as sugarcane bagasse, corn stover biomass and lignocellulosic energy
crops have already been successfully used as the major feedstock for the purpose
(Datar et al., 2007, de Vrije et al., 2009, Pattra et al., 2008). However, most of the waste
materials and lignocellulosic materials, in particular require proper pre-treatment prior to
fermentation (Abreu et al., 2012).
Crude glycerol (CG) generated during biodiesel manufacturing process is a readily
usable substrate for biohydrogen production. Actually, during biodiesel production
(trensesterification), for every 100 Kg biodiesel, 10 Kg of CG is generated as byproduct
(Chatzifragkou et al., 2012, Santibáñez et al., 2011). Owing to its crude nature, CG
does not have any significant commercial application and further stricter environmental
regulations
are
applied
for
its
(http://www.epa.gov/region7/priorities/agriculture/pdf/biodiesel_manual.pdf
disposal
(accessed
on 28_08_2012)). Therefore, considering the increase in global biodiesel production,
cost effective management of large amounts of byproduct (CG) could be an emerging
challenge for biodiesel manufacturers. Hence, CG bioconversion for hydrogen
production could be an alternative option for its sustainable management. Meanwhile,
CG is mainly a carbon source for industrial fermentation and hence, additional nutrient
sources are generally applied for its improved bioconversion (Liu et al., 2011, Ngo et al.,
2011, Wilkens et al., 2012). However, according to an analysis, the cost of the
supplementary nutrients used for H2 production by CG bioconversion may be as high as
256
82 % of total process cost (Sarma et al., 2013a). Therefore, the major objective of the
present investigation is to evaluate different less expensive nutrient sources for potential
application as a replacement of costly synthetic media components required for the
process. H2 production could be realized either by dark fermentation or by photofermentation. However, owing to its higher hydrogen production rate, dark fermentation
is considered more advantageous over photo-fermentation (Ciranna et al., 2011) and
hence, dark fermentation has been considered for the present investigation. Similarly,
Enterobacter
aerogenes,
well-known
facultative
anaerobic
bacterium
for
CG
bioconversion (Ito et al., 2005, Marques et al., 2009, Sakai et al., 2007), has been used
in the present study. Moreover, increased hydrogen partial pressure in the headspace
of the reactor is known to be thermodynamically unfavorable for H2 synthesis and
metabolic shift towards the production of more reduced end products could be a
consequence (Ciranna et al., 2011, Levin et al., 2004). Therefore, the present
investigation was carried out at relatively low H2 partial pressure by periodically
removing the produced H2 from the reactor headspace.
Materials and Methods
Microorganism and inoculum development
Enterobacter aerogenes NRRL B 407 (ARS, USDA, USA), a gram-negative, facultative
anaerobic, rod-shaped bacterium belonging to the family Enterobacteriaceae was
considered for the present investigation. A nutrient medium composed of glucose (5
g/L), casein peptone (5 g/L), KH2PO4 (2 g/L), yeast extract (0.5 g/L) and MgSO4.7H2O
(0.5 g/L) (Beckers et al., 2010) was used to maintain the microorganism (21 ± 1°C) and
the same medium was also used for inoculum development throughout the
investigation. For both purposes, 125 mL serum bottles (working volume 50 mL) were
used and the media was sparged with ultrapure N2 gas for 2 min to create anaerobic
environment for supporting the growth of the microorganisms. Following N2 sparging,
immediately the bottles were sealed with aluminum crimp seal containing silicone
septum (Fisher scientific, Canada) by using a hand operated crimper (E-Z Crimper™,
VWR, Canada). Prior to inoculation, the bottles were autoclaved and cooled to room
257
temperature. Inoculation was made by using a sterile syringe which was followed by
incubation of the bottles in orbital incubator shaker maintained at 150 rpm and 30±1°C
(Marques et al., 2009). Freshly prepared culture media containing the microorganism in
its exponential growth phase was used as inoculum (5%, v/v) for all the experiments
conducted in this investigation.
Chemicals and other materials
Chemicals, such as glucose, urea, MgSO4 7H2O, NaOH, sodium periodate, acetone,
acetylacetone, bromophenol blue, HCl, ethanol and acetic acid were purchased from
fisher scientific, Canada. Casein peptone and KH2PO4 were purchased from VWR,
Canada and the yeast extract was a kind gift from Lallemand, Canada. Crude glycerol
(CG), a by-product of biodiesel production by transesterification was collected from
Rothsay®, Canada and used as the major feedstock for present study. Rothsay mainly
produces biodiesel and CG by using the waste materials from meat processing plants
and used grease from restaurants (http://www.rothsay.ca/products/biodiesel/; (accessed
on 06-08-2012)). Secondary sludge (SS) used in the present investigation was collected
from Quebec urban community (CUQ) wastewater treatment plant, Quebec, Canada
(DP Mohapatra et al., 2012). Brewery liquid waste (BLW) used in the present
investigation was kindly provided by La Barberie (Quebec), Canada. The slaughter
house liquid (SL) waste material was collected from Gibiers Canabec, Quebec, Canada.
It was mainly composed of blood and other liquid waste of meat processing operation,
which was used without any pretreatment. Similarly, starch industry wastewater (SIW)
from ADM Ogive; Candiac, Quebec, Canada and the apple pomace sludge (AS) was
obtained from Lassonde Inc., Rougemont, Montreal, Canada (see reference for detailed
characterization) (Dhillon et al., 2011).
Characterization of crude glycerol
As specifically described in later section, glycerol content of the CG sample (Sarma et
al., 2013b) was determined by spectrophotometric method. To determine the total
suspended solids, the CG sample (10 g) was diluted with 1 L of deionized water and
passed through a glass fiber filter paper (G6, Fisher, Canada). Finally, the filter paper
258
was dried in oven (105 ±1 °C to constant weight) to remove the water and weighed after
cooling.
From the difference of filter paper weight before and after filtration, total
suspended solids were calculated and the result was expressed as mg/Kg.
Soap present in CG was determined by titration and expressed as sodium oleate.
Briefly, a mixture of 60 mL acetone and 0.15 mL of 0.5 % (w/v) bromophenol blue (in 95
%
ethanol)
was
prepared
and
neutralized
with
0.1
M
NaOH
(www.oiv.int/oiv/files/CODEX_2012_EN.pdf (accessed on 11_09_2012)). The solution
was mixed with 10 g of CG sample and heated in a water bath (70 ±1°C) for 1 min.
Later, the mixture was titrated using hydrochloric acid solution (0.1M). On complete
neutralization of the soaps present in the CG sample by the acid used, blue color of the
solution (pH indicator) disappeared. Further, the amount of the acid needed for the
experiment was recorded and used to calculate the soap content of the CG sample.
Hydrogen production experiments
As already outlined for inoculum development, 125 mL serum bottles (working volume
50 mL) were used as anaerobic bioreactor for hydrogen production experiments. Other
conditions of media preparation and further incubation were kept the same. In a
previous investigation, different initial concentrations of CG have been evaluated and 10
g/L CG was found to be optimum for bio-hydrogen production (unpublished result).
Therefore, for present investigation, 10 g/L CG (CG diluted with deionized water) was
used as the basic media for all hydrogen production experiments, which was
supplemented with different nutrient sources according to the demand of the
investigation (as described below). Further, pH 6.0 is reported to be the optimum for
hydrogen production by E. aerogenes, the microorganism used in the present
investigation (Sakai et al., 2007). Hence, all the experiments were carried out at pH 6.0.
Similarly, all the experiments were carried out using 5 % (v/v) aforementioned inoculum,
which was equivalent to 0.25 mg dry cell mass. At 12 h interval, 1 mL of the aqueous
media was collected by sterile syringe and pH and residual glycerol concentration was
measured. Accordingly, the composition of produced gas mixture was analyzed by gas
chromatography and the volume of produced H2 was measured at 12 h interval and
259
cumulative H2 production was expressed as millimoles per liter (mmol/L) (see below).
Reported H2 concentrations are the average of the readings from duplicate
experiments.
Evaluation of organic waste materials as supplementary nutrient source for CG
bioconversion
In the present investigation, a range of waste materials, such as secondary sludge (SS)
from waste treatment plant, brewery liquid waste (BLW), starch industry wastewater
(SIW), apple pomace sludge (AS) and slaughterhouse liquid (SL) wastes have been
evaluated as a supplementary nutrient source for improved H2 production by CG
bioconversion. No pretreatment was made (unless specifically mentioned) for such
waste materials prior to their use in media preparation. The pH, total solids, total organic
carbon and ammoniacal nitrogen content of the sludge sample used in the present
investigation were 6.19±0.2, 17.46±2.3 (g/L), 282±10 (g/L) and 0.17±11 (g/L),
respectively. Detailed characterization of the wastewater sludge could be found in
Mohapatra et al, 2011 (D.P. Mohapatra et al., 2011). Similarly, physical properties and
elemental composition of SIW and AS considered for present investigation could be
found in Dhillon et al, 2011 (Dhillon et al., 2011). To investigate the effect of these
materials on H2 production using separate serum bottles, SS (10, 20, 40 and 80 mg /L),
SIW (0.2, 1, 3 and 5 %, v/v), AP (10, 20, 100, 300 and 500 mg/L) and SL (10, 20, 100,
300 and 500 mg/L) were mixed with 10 g/L CG and the resulting media types were
autoclaved. All the other conditions of media preparation, inoculation, incubation and
sample collection were the same as discussed in previous section.
In the case of brewery waste, it mainly contains yeast biomass and liquid material left
after product recovery. Two separate investigations were carried out to evaluate the
wastes for H2 production. For the first set of experiments, 10 g/L CG was separately
mixed with 20, 60, 120 and 240 mg/L of the liquid waste (BLW) and autoclaved.
Alternatively, for second set of experiments, the waste was mixed with distilled water to
have a final concentration of 100 g/L. Resulting mixture was centrifuged at 5000 x g for
5 min, the supernatant was discarded and the solid fraction containing biomass was
260
collected. The purpose of this step was to remove the alcoholic component that may be
present in the brewery waste and could be inhibitory for microbial growth. However, it
was an assumption and no experiment was conducted to verify the necessity of this
step. Thus, the biomass collected (BWB) after centrifugation was supplemented with 10
g/L CG at different concentrations ranging from 10 to 160 mg/L, autoclaved and
hydrogen production experiment was carried out as outlined in previous sections.
Evaluation of urea as supplementary nitrogen source for CG bioconversion
This investigation was conducted to determine the potential of urea as a less expensive
nitrogen source for H2 production by CG bioconversion and six different concentrations
of urea ranging from 10 to 180 mg/L have been evaluated. As already mentioned for
different waste materials, 10 g/L CG was enriched with different concentrations of urea,
pH was adjusted, sparged with N2 (see above) and separately autoclaved. All the
conditions of incubation, sample collection and analysis were similar as already
described.
Analytical methods
Glycerol
Glycerol concentration of the aqueous samples collected during hydrogen production
experiments were measured by the spectrophotometric method (Bondioli et al., 2005).
The method is based on periodate oxidation of glycerol followed by optical density
measurement at 410 nm. In practice, 1 mL of the aqueous sample was mixed with 1 mL
of ethanol (47.5% v/v) followed by addition of 1.2 mL each of acetylacetone solution (0.2
M) and sodium periodate solution (10 mM). Subsequently, using a water bath shaker,
the reaction mixture was incubated at 70 ± 1 °C for 1 min and immediately cooled to 2025 °C by using cold water. The samples were maintained at the same temperature until
analyzed by UV-Vis spectrophotometer (Carry 100 Bio®, Varian USA) (Sarma et al.,
2013a).
261
Hydrogen analysis
H2 and CO2 are principal gases produced during CG bioconversion (Marques et al.,
2009) and H2 in the mixture was analyzed by a gas chromatograph (Varian 3800, USA)
fitted with PoraPLOT Q® column (Agilent technology, USA) and thermal conductivity
detector (TCD). The column temperature was fixed at 100 °C and nitrogen was used as
the carrier gas. The gas flow rate was maintained at 3.5 mL min-1 with a retention time
(H2) of 4.5 min. As described by previous investigators, the volume of produced H2 was
measured by the inverted cylinder –NaOH (10%) method (Junyapoon et al., 2011,
Maeda et al., 2008). Briefly, to remove CO2 fraction from the gas mixture, the produced
gas was passed through 100 mL NaOH (10% w/v). Subsequently, it was collected in a
water filled inverted graduated glass cylinder placed in a partially filled (with water)
beaker. Based on displacement of water level in the cylinder, volume of produced H2
was calculated (Junyapoon et al., 2011, Maeda et al., 2008) and, where applicable,
considering the temperature and atmospheric pressure, volume of produced gas was
expressed as millimoles/L media (http://www.easycalculation.com/chemistry/idealgas.php (accessed on 16_07_2012).
Crude glycerol characterization
The CG fraction considered for the present investigation contains 23.63 % (w/w)
glycerol and it has a pH of 3.4 (at 24 ± 0.5 °C) (Sarma et al., 2013b). In addition, it was
found to contain 300 mg suspended solids per Kg of CG. Similarly, the soap content of
the CG sample was found to be 0.364 ± 0.05 % (w/w). This value is significantly lower
than 20-31 % (w/w) of soap reported for different CG samples (Hu et al., 2012).
Considering relatively low pH (3.4) of present CG sample, such observation may be due
to possible acidification of soap. Meanwhile, soap has potential inhibitory effect on
bacterial
growth
(http://www.enterprise-europe-
network.ec.europa.eu/src/matching/templates/completerec.cfm?bbs_id=172911
(accessed on 11_09_2012)). Therefore, CG with negligible soap content could be
considered ideal for bioconversion purposes, such as hydrogen production.
262
Evaluation of different waste materials as nutrient supplements
CG is mainly a carbonaceous material and hence, addition of supplementary nutrient
source could have improved its potential as a feedstock. In fact, improve bioconversion
efficiency has been observed by addition of supplementary nutrient sources, such as
peptone, yeast extract and a range of minerals (micro and macro nutrients) (Fernandes
et al., 2010, Ito et al., 2005, Ngo et al., 2011, Sakai et al., 2007, Selembo et al., 2009).
However, most of the synthetic or semisynthetic ingredients have commercial
importance and hence are expensive. Further, production of such compounds, similar to
any other manufacturing processes might have potential negative effect on the
environment. Therefore, extensive application of such compounds for environmentally
sustainable management of CG may not be a feasible concept. On the contrary, a
range of agro-industrial organic waste materials could be a suitable alternative of such
synthetic compounds. Further, this strategy not only has the potential to replace the
expensive synthetic materials from the process, but also could be a mean for
sustainable management of different agro-industrial wastes.
For this purpose, different nutrient rich waste materials, ranging from secondary sludge
of municipal wastewater treatment plant to wastewater from slaughter house have been
evaluated. Commonly, these materials are rich in proteinaceous compounds and
minerals and should be a good source of nutrients for the proposed process. However,
considering their crude nature, inhibitory effect of such compounds could not be ruled
out and hence, each of them was separately evaluated. Batch H2 production
experiments were conducted where 10 g/L CG was supplemented with different
concentrations of these materials and cumulative H2 production was considered as the
measure of their potential application. Moreover, an experiment was carried out where
cumulative H2 production using 10 g/L CG (without any supplementary component) was
reported (Sarma et al., 2013b). This result was considered as the negative control and
all results from subsequent investigations were compared with this value to quantify the
improvement in H2 production (see below).
263
Figure 3.2.1(a) shows the H2 production profiles obtained for different concentrations of
slaughter house liquid waste used as a supplementary nutrient. Cumulative H2
production, after the period of the experiment was found to be almost similar for 10-100
mg/L SL; where, 10 mg/L gave the highest cumulative production. Further increment in
SL concentration (300-500 mg/L) was found to be highly inhibitory for the process.
Similar response trend was found for apple pomace sludge (AS) too (Figure 3.2.1 (b).
Higher production was obtained for the concentration range of 10-20 mg/L, whereas, at
relatively higher concentrations (300-500 mg/L) cumulative H2 production was
diminished.
Further, Figure 3.2.1 (c) shows the H2 production profiles for different concentrations of
SS supplemented to the process. Similar to the previous cases, SS in the range of 1080 mg/L was showing nearly similar response in terms of H2 production and at relatively
higher concentration (500 mg/L and beyond), H2 production was considerably inhibited
(data not shown). Figure 3.2.1 (d, e and f) show the profiles obtained for BLW, BWB
and SIW, respectively. Overall, for all the waste materials tested in the present study,
relatively lower concentration gave better response and was found to be inhibitory at
higher concentrations. Further, as shown in Figure 3.2.2, best response from each
independent investigation was compared with response from a controlled experiment
(10 g/L without any supplementary component) (Sarma et al., 2013b). From Figure
3.2.2, it could be concluded that SS, SL, BLW and BWB (at relatively low concentration)
were found to improve the cumulative H2 production over the control condition. Among
these materials, BWB (10 mg/L) was the most successful supplement. As obvious from
Figure 3.2.2, BWB was able to improve the cumulative H2 production by 27.30 ± 3.54 %.
Similarly, SL was the other material to improve the production by 18.81 ± 3.56 %.
Actually, brewery and slaughterhouse wastes are nitrogen rich (proteinaceous)
materials. Hence, improved H2 production could be attributed to improved cell
proliferation in the presence of nitrogenous compounds. However, these wastes were
also found to inhibit H2 production when used at relatively higher concentration (usually
more than 100 mg/L) (Figure 3.2.1 (a). The possible reason for such observation may
be that the addition of these wastes at higher concentration could decrease the C-N
264
ratio of the broth leading towards diminished H2 production (Lin et al., 2004). Similarly, a
metabolic pathway shifting in the presence of relatively higher concentration of these
wastes (BWL, BWB, and SL) resulting in increased production of reduced end-products
and decreased H2 yield could not be ignored. However, it is only a possibility and for
further explanation, detailed investigation could be conducted to get the information on
the types as well as amounts of different fermentation end-products. On the contrary,
AP and SIW were unable to impart any improvement in H2 production at any of the
tested concentrations (Figure 3.2.2). Considering the crude nature of all the materials
evaluated in the present study, presence of growth inhibitors (such as, heavy metals)
could be the reason for their negative effect. Similarly, shifting of metabolic pathway
may be the other most possible reason. Further, in the special cases of AP and SIW,
their non-proteinaceous origin (unlike BWB, SL and SS) may be a reason for their
failure to improve the CG bioconversion process even at lower concentrations.
Most of the reports presently available on CG bioconversion and H2 production are
summarized in Table 3.2.1. As it is obvious from the Table 3.2.1, a range of synthetic
media components were used in all such studies. Further, to maintain the process pH
during all the H2 production studies, either commercial buffering agents were used or
automatic pH controlling system applied. However, the application of any such synthetic
media component can increase the process cost by up to 82 % (Sarma et al., 2013a).
On the contrary, in the present investigation the synthetic media components were
successfully replaced by waste materials, such as BWB and SL. Similarly, no buffering
agent was used for maintaining a constant pH during the process. Still, as obvious from
Table 3.2.1, the results are highly comparable with any available report on H2
production by CG bioconversion. Unlike, conventional pure substrate based CG, special
chemical composition of the CG (meat processing and restaurant waste based) used in
present investigation (Sarma et al., 2013b), its inherent buffering property as well as
various supplementary nutrients could be attributed to observed better productivity.
Thus, the present finding has depicted that CG supplemented with small amount of (1020 mg/L) nitrogen rich waste (especially BWB) could be as effective as a process
augmented with synthetic media (~5-6 g/L) components (Table 3.2.1).
265
As summarized in Figure 3.2.2, glycerol utilization and H2 production (maximum)
profiles obtained for different investigations was found to have good correlation with
media pH recorded at the end of different processes. Compared to the control
experiment (without any supplement), the investigations carried out with BWB and SL
were found to have lower final broth pH. Such observation could be due to increased
acetic acid production corresponding to enhanced H2 production in those cases (Van
Ginkel et al., 2005). Further, corresponding to relatively low glycerol utilization for SIW
and AP (61 ± 3.3 and 72.1 ± 3.4 % respectively; Figure 3.2.2), final media pH in those
cases were higher than the cases of any other supplements. Most importantly, the strain
(E. aerogenes NRRL-B407) used in the present study was found to produce H2 at a pH
well below 4 (around 3.3). Such finding could be beneficial, as in the case of dark
fermentative H2 production the process (batch) is usually terminated by pH change due
to organic acid production. Hence, improved H2 production could be expected by
application of this acid tolerant strain.
Effect of urea on H2 production using CG
Similar to different waste materials, urea was also evaluated as a possible low cost
nitrogen source for CG bioconversion. Figure 3.2.3 shows the H2 production profiles
obtained for different concentrations of urea supplemented to the process. From Figure
3.2.3, it could be summarized that among different concentrations of urea, maximum
cumulative H2 production was obtained for 10 mg/L urea. Further, H2 production was
found to be diminished with the increase in urea concentration as well as, a prominent
lag phase (in H2 production) was observed at relatively higher concentration (180 mg/L
urea). Similar to the case of different nitrogen rich waste materials, a possible reason for
such observation may be that with the increase in urea concentration, the C-N ratio of
the culture media decreased and H2 production has been repressed (Kapdan et al.,
2006, Rojas et al., 2010). Meanwhile, at relatively low urea concentration (10 mg/L), H2
production was found to be improved by 38.57 ± 3.66 % (Figure 3.2.2). As already
mentioned, different reports on H2 production by CG bioconversion have been
summarized in Table 3.2.1. It could be observed from the table (Table 3.2.1) that the
performance of the present process is highly comparable with different previous
266
investigations. Further, the significance of the present observation is that a small
amount of urea (10 mg/L) was successfully used to replace around 5-6 g/L of costly
synthetic media components (nutrients and buffering agents) without compromising the
H2 production efficiency of the process (Table 3.2.1). Hence, reduced process cost
could be an advantage of the present process over different presently known equally
successful processes (Table 3.2.1).
Thus, less expensive nutrient sources such as SL, BWB and urea could be considered
as potential additive for sustainable bio-refinery of CG. Most importantly, application of
slaughterhouse liquid waste for improved bioconversion of meat processing waste
based biodiesel derived CG has particular significance as both the industries are
interlinked. Hence, waste generated in one process could be sustainably applied for
waste valorization of the other. Based on this concept and considering proteinaceous
nature of SL, its possible application in inoculum development (preparation of protein
rich medium) for CG bioconversion process could be considered as a promising option
for future investigation.
Conclusions
Bioconversion of biodiesel byproduct (CG) is a relatively new and promising process to
be introduced for hydrogen production. However, the process is highly limited by
prohibitory process cost attributed by expensive supplementary media components. In
the present investigation, waste materials, such as BWB, SL, SS, AP, SIW and urea,
less expensive nitrogen sources, have been evaluated as the replacement of expensive
media components. E. aerogenes has been used for the study where, 10 g/L CG was
the sole carbon source and it was observed that addition of SL, BWB and urea can
enhance the production by 18.81 ± 3.56, 27.30 ± 3.54 and 38.57 ± 3.66 %, respectively.
Similarly, cumulative production of 116.41 ± 3.72 mmol H2/L media and a maximum H2
production rate of 965 ± 35 mL/L/d have been achieved by using urea as low as 10
mg/L as the supplementary nitrogen source. Present findings were found to be
comparable to any other reports available on CG bioconversion, demonstrating the
267
suitability of BWB, SL and urea as the replacement of expensive media component
used for the process.
Acknowledgments
The authors are thankful to Mr. Sebastien Duval for his assistance in gas
chromatographic analysis. Financial assistance from Natural Sciences and Engineering
Research Council of Canada (Discovery Grant 355254), Ministère des Relations
internationales du Québec (coopération Paraná-Québec 2010-2012) and Le Centre de
recherche industrielle du Québec (CRIQ), MAPAQ (No. 809051) has also been
sincerely acknowledged.
268
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Table 3.2. 1: The summary of different reports available on H2 production by CG bioconversion
Sl.
no
Microorganism
Inoculum
size
CG
concentration
(g/L)
CG origin
Additional
nutrient
pH
controlled/
buffering
agent added
H2 production
1
Enterobacter
aerogenes NBRC
12010
4% (v/v)
CG containing
9.9 g/L glycerol
used food
oils
Synthetic
media
yes
138.6 mmol/L
(Sakai et al.,
2007)
2
Enterobacter
aerogenes ATCC
13048
0.1 g/L
(biomass)
20
-
Synthetic
media
yes
2500 mL/L
(Marques et
al., 2009)
3
Enterobacter
aerogenes HU101
4%
CG containing
1.7 g/L glycerol
-
Synthetic
media
yes
20.67 mmol/L*
(Ito et
2005)
4
Mixed culture
10 % (v/v)
COD 3.6
Vegetable
oil
Synthetic
media
yes
200 mL/g COD
(Fernandes
et al., 2010)
5
Thermotoga
neapolitana
10 % (v/v)
05
-
Soybean flour
yes
518.25 ± 25.9 mL/L
(Ngo et al.,
2011)
6
Mixed culture
1g/L (soil)
03
Soybean oil
Synthetic
media
yes
7.018 mmol/L*
(Selembo et
al., 2009)
7
E. aerogenes
NRRL B407
5% (v/v)
10 (2.3 g/L
glycerol)
Meat
processing
and
restaurant
Slaughter
house liquid
waste (10
mg/L)
No
2400 mL/L (99.78
mmol/L)
Present
study
273
*
Ref.
al.,
8
9
waste
Brewery waste
biomass (10
mg/L)
2570 mL/L (106.84
mmol/L)
Urea (10 mg/L)
2800 mL/L (116.41
mmol/L)
274
(a)
6
5
4
3
2
SL 10 mg/L
SL 20 mg/L
SL 100 mg/L
SL 300 mg/L
1
0
0
24
48
72
96
Time (h)
275
120
144
168
192
(b)
4.5
4
3.5
3
2.5
2
AS 10 mg/L
AS 20 mg/L
AS 100 mg/L
AS 300 mg/L
AS 500 mg/L
1.5
1
0.5
0
0
24
48
72
96
Time (h)
276
120
144
168
192
(c)
6
5
4
3
2
SS 10 mg/L
SS 20 mg/L
SS 40 mg/L
SS 80 mg/L
1
0
0
24
48
72
96
Time (h)
277
120
144
168
192
(d)
6
5
4
3
2
BWL 20 mg/L
BWL 60 mg/L
BWL 120 mg/L
BWL 240 mg/L
1
0
0
24
48
72
96
120
Time (h)
278
144
168
192
(e)
6
5
4
3
BWB 10 mg/L
BWB 20 mg/L
BWB 40 mg/L
BWB 80 mg/L
BWB 160 mg/L
2
1
0
0
24
48
72
96
Time (h)
279
120
144
168
192
(f)
3
2.5
2
1.5
SIW 0.2 % v/v
SIW 1 % v/v
SIW 3 % v/v
SIW 5 % v/v
1
0.5
0
0
24
48
72
96
120
144
168
192
Time (h)
Figure 3.2. 1: H2 production profiles obtained for different concentrations SL (a), AS (b), SS (c),
BWL (d), BWB (e) and SIW (f) supplemented to 10 g/L CG
280
120
Glycerol
110
pH
H2 production (mmol/L);
Glycerol utilization (%)
3.9
Hydrogen
3.8
3.7
100
3.6
90
3.5
80
3.4
70
Final pH of the broth
130
3.3
60
3.2
50
40
3.1
Urea 10
mg/L
BWB 10 SL 10 mg/L SS 20 mg/L BWL 20 AP 10 mg/L SIW 1%
mg/L
mg/L
(v/v)
CG 10g/L
(control)
Figure 3.2. 2: Maximum cumulative H2 production (mmol/L), glycerol utilization (%) and broth pH
recorded at the end of fermentation for different material supplemented to 10 g/L CG. The control
indicates the results obtained for 10 g/L CG without any additive.
281
7
Urea 10 mg/L
6
Urea 20 mg/L
5
Urea 60 mg/L
4
Urea 120 mg/L
Urea 180 mg/L
3
2
1
0
-1
0
24
48
72
96
120
144
168
192
Time (h)
Figure 3.2. 3: H2 production profiles obtained for different concentrations urea supplemented to 10
g/L CG
282
CHAPTER 4
EFFECT OF CRUDE GLYCEROL IMPURITIES: SPECULATIONS AND
FACTS
Chapter 4. Effect of crude glycerol impurities: speculations and facts
PART I
INVESTIGATION OF THE EFFECT OF DIFFERENT CRUDE GLYCEROL
COMPONENTS ON HYDROGEN PRODUCTION BY ENTEROBACTER
AEROGENES NRRL B-407
Saurabh Jyoti Sarmaa, Gurpreet Singh Dhillona, Satinder Kaur Brara, Yann Le
Bihanb, Gerardo Buelnab, Mausam Vermac
a
b
c
CO2 Solutions Inc., 2300, rue Jean-Perrin, Québec, Québec G2C 1T9 Canada
[email protected])
Renewable Energy 60 (2013) : 566–571
285
Résumé
Le glycérol brut (GB) est le principal sous-produit (déchets) de la production de
biodiesel (par transestérification de lipides) et en poids, il pourrait être aussi élevé que
10% (poids/poids) du produit final. Considérant l'intérêt mondial pour la production de
biodiesel, il existe un besoin pour une gestion durable de ces déchets. Dans ce
contexte, la production de biohydrogène par l'utilisation du GB pourrait être une option
intéressante. De plus, l'hydrogène est considéré comme étant la plus propre des
énergies renouvelables pour le remplacement des combustibles fossiles. Cependant, le
GB contient quelques impuretés, tels que le méthanol, le NaCl les savons et leurs effets
individuel et interactif sur la production d'hydrogène doivent être examinés. Ainsi, cette
étude traite de l'effet des contaminants du GB tels que le méthanol, le NaCl et le savon
sur la production de biohydrogène. Sur la base de la vérification effectuée en utilisant la
méthodologie de surface de réponse, on a observé que le savon est le facteur le plus
significatif (valeur p = 0,0104 ˂ 0,05) pour inhiber la production d'hydrogène. De façon
similaire, le méthanol a un effet négatif sur la production d'hydrogène dans la gamme
de concentration de 1.3 à 3.3 g/L. En contrepartie, le NaCl à des concentrations de 0.15
à 0.55 g/L a montré un effet positif sur le processus de bioconversion. Dans l'ensemble,
la présence des différents contaminants contenus dans le GB a eu un effet inhibiteur
réduisant la production cumulative de H2 de 21,6 à 95,3%. En outre, sur la base des
observations présentes une nouvelle stratégie intégrée pour l'enlèvement du savon et la
gestion du pH lors de la fermentation a été émise afin d'améliorer la production de H2 à
partir du GB.
Mots-clés: biodiesel; biohydrogène; glycérol brut; impureté; savon
286
Abstract
Crude glycerol (CG) is the main by-product (waste) of biodiesel production (by
transesterification of lipids) and by weight it could be as high as 10 % (w/w) of the final
product. Considering global interest for biodiesel production, there is a need for
sustainable management of the waste. In this context, bio-hydrogen production by
utilization of CG could be an interesting option. Moreover, hydrogen is considered as
the cleanest renewable alternative of fossil fuels. However, CG contains a few
impurities, such as methanol, NaCl and soap and their individual and interactive effect
on hydrogen production should be investigated. Thus, present study deals with the
investigation of the effect of methanol, NaCl and soap on hydrogen production and
substrate (glycerol) utilization. Based on the investigation carried out using response
surface methodology, it was observed that soap is the most significant factor (p value =
0.0104 ˂ 0.05) to inhibit hydrogen production. Similarly, methanol was also found to
have negative effect on hydrogen production in the concentration range (1.3 to 3.3 g/L)
considered. Meanwhile, NaCl (0.15- 0.55 g/L) showed positive effect on the process.
Overall, presence of different impurities was found to reduce the cumulative H2
production by 21.56 to 95.31 %. Furthermore, based on present observations a novel
integrated strategy for soap removal and process pH management has been
hypothesized for improved H2 production by CG bioconversion.
Keywords: biodiesel; bio-hydrogen; crude glycerol; impurity; soap
287
Introduction
Shrinking fossil fuel reserves, the concern of greenhouse gas emission and global
climate change, strict regulations for mandatory renewable content in transportation
fuels as well as subsidies offered by different governments are some of the reasons for
wide exploration of biodiesel production technologies. Transesterification of vegetable
oils, animal fats and other lipids (microbial origin) is the core process involved in
biodiesel production. During trans-esterification process, crude glycerol (CG) is
generated as a waste by-product, which is around 10 % by weight of the biodiesel
produced by the process (Chatzifragkou et al., 2012). Considering possible increase in
global biodiesel production and the amount of CG produced by the process, there is a
need for a sustainable strategy of CG management. Utilization of CG as a feedstock for
microbial production of commercially important products could be an interesting option
(Dobson et al., 2012). In this context, production of bio-hydrogen by bioconversion of
CG has been evaluated by various researchers (Sabourin-Provost et al., 2009, Sakai et
al., 2007, Selembo et al., 2009). Actually, H2 is considered as an ideal renewable
energy source for future because it does not produce any greenhouse gas during
combustion. In fact, it is the only carbon free fuel to produce only water as combustion
by-product as well as it has highest energy content among gaseous fuels of equivalent
weight (Meher Kotay et al., 2008). Similarly, it can be easily fed to fuel cells or internal
combustion engines for energy conversion (Fang et al., 2006). However, CG is not a
pure substrate for hydrogen production and it may contain a range of impurities.
Methanol, salt and soap are three of the major impurities present in CG and they might
have certain effect on hydrogen production by CG bioconversion (Venkataramanan et
al., 2012, Yang et al., 2012). In fact, type of substrate and carbon-nitrogen ratio of the
media has significant influence on hydrogen production (Zhao et al., 2008). As the
impurities such as methanol and soap could also be utilized as carbon sources, their
presence or absence in the media might affect the performance of the process.
Moreover, all three impurities might show certain interactions among themselves and
these possibilities are yet to be explored for a bio-hydrogen production process. Thus,
288
the main objective of the present study was to evaluate the effect of these three CG
impurities on hydrogen production by glycerol bioconversion.
The investigation has been carried out using response surface methodological
approach and the experimental plan was prepared by using central composite design.
Batch bio-hydrogen production experiments were carried out using small scale
anaerobic bioreactors by employing Enterobacter aerogenes (Nakashimada et al.,
2002, Rachman et al., 1997).
Chemicals
Crude glycerol (CG) used in the present investigation is a by-product of biodiesel
production process which was collected from Rothsay®, Canada. Other chemicals, such
as glucose, ethanol, NaOH, HCl, MgSO4. 7H2O, sodium periodate, acetone,
acetylacetone, bromophenol blue and acetic acid were purchased from Fisher scientific
(Missisauga, Ontario, Canada). Casein peptone and KH2PO4 were supplied by VWR
International (Ontario, Canada) and the yeast extract used in the present study was a
gift from Lallemand, Canada.
Microorganism and inoculum preparation
Enterobacter aerogenes NRRL B 407, the microorganism considered for the present
investigation was provided by ARS, USDA, USA. It is a gram-negative, facultative
anaerobic, rod-shaped bacterium known to produce hydrogen by using a range of
organic substrates. The microorganism was grown and maintained in a culture medium
composed of glucose (5 g/L), casein peptone (5 g/L), KH2PO4 (2 g/L), MgSO4 ̇ 7H2O
(0.5 g/L) and yeast extract (0.5 g/L) (Beckers et al., 2010). For the entire investigation,
the same growth medium was used to prepare the inocula. For inoculum development,
the microorganism was anaerobically grown at 30 ±1° C (150 rpm) using a rotary shaker
incubator and 5% (v/v) freshly prepared (exponential growth phase) cell suspension
was used as inoculum for all the experiments conducted in the present investigation.
289
Saponification of crude glycerol and separation of soap
The soap used in the present investigation was separated from a CG sample collected
from biodiesel production plant. The CG sample was first saponified by a slightly
modified method based on the original method reported by (Hu et al., 2012). Briefly, 10
g CG was dissolved 100 mL water and the solution was mixed with 100 mL of 50 g/L
NaOH-ethanol solution. The mixture was taken in a screw cap bottle and kept in a rotary
shaking water bath (90 ± 1°C/50 rpm) for 1h. After cooling the mixture to room
temperature equal volume of deionized water was added and the soap present in the
solution
was
precipitated
by
salting
out
http://people.cedarville.edu/employee/gollmers/gsci1020/labs/08g1020lab5.pdf
(accessed on 29/11/2012) (using 10% w/v NaCl). Precipitated soap was separated from
the solution by centrifugation (5000 g for 20 min) and lyophilized for 24h prior to its
application in the investigation. Entire process of soap separation has been shown in
Figure 4.1.1.
Experimental design and hydrogen production experiments
As already mentioned, methanol, soap and NaCl are the three impurities of crude
glycerol which were evaluated in the present investigation for their role in CG
bioconversion and hydrogen production. A central composite design was employed to
study their effect using response surface methodology (RSM). In Table 4.1.1, the
amount of the impurities which could be present in a CG sample has been summarized
based on literature data. Similarly, 10 g/L CG has been found to be optimum for
biohydrogen production (Sarma et al., 2013). Therefore, as presented in Table 4.1.1
and 2, the minimum concentrations of the impurities that could be present in 10 g/L CG
have been considered as the central points of the independent variables (impurities) for
present experimental design. Further, the experimental design has been constructed by
using Design-Expert® -7 software (Stat-Ease Inc. Minneapolis, MN) and the design has
been extended up to + α and – α level (Table 4.1.2). Based on the design, 20 different
runs of experiments were performed using different combinations of the independent
variables (impurities) as presented in Table 4.1.3. Cumulative hydrogen production
290
(mmol/L after 48h) and percent utilization of glycerol have been considered as the two
responses (dependent variables) of the investigations (Table 4.1.3).
All the experiments were carried out by using 125 mL serum bottles with 50 mL working
volume. The experiments were performed using (separate serum bottles) a culture
media composed of pure glycerol (5 g/L), casein peptone (1 g/L) and yeast extract (0.05
g/L) with which different concentrations of methanol, soap and NaCl were mixed
according to the experimental plan (Table 4.1.3). One control experiment was carried
out using the same media composition (but devoid of any impurity) and this experiment
was not a part of the present experimental design. Further, pH 6 was found to be
optimum for H2 production by E. aerogenes (Sakai et al., 2007). Hence, the pH of the
culture media taken in different serum bottles was adjusted to 6 by using 0.1 N
NaOH/HCl solutions. Subsequently, the media were sparged with ultrapure N2 gas for 2
min to create anaerobic environment for supporting the growth of the facultative
anaerobic bacteria (E. aerogenes). The bottles sparged with N2 were immediately
sealed with aluminum crimp seal containing silicone septum (Fisher scientific, Canada)
with the help of a hand operated crimper (E-Z Crimper™, VWR International, Ontario,
Canada). Sealed bottles were sterilized by autoclaving and cooled to room temperature
following which they were inoculated using the inoculum mentioned in previous section
(section 2.2). Inoculation was made through the silicone septum of the crimp seal of the
serum bottle by using a sterile syringe fitted with a fine needle. Finally, the bottles were
incubated at 30±1°C by using an orbital incubator shaker maintained at 150 rpm (Sarma
et al., 2013). By using a 5 mL gastight syringe gas samples (1 mL each) were collected
from the headspace of the culture bottle and kept in gas sampling vial (Exetainer®,
Labco, UK) for gas chromatographic analysis. Similarly, at the end of the fermentation
(H2 production process), 1 mL of liquid sample was also collected from each bottle for
residual glycerol content analysis.
291
Analytical techniques
Hydrogen analysis
After 48h of incubation, H2 gas produced in the case of each experiment was collected
(as already mentioned in previous section) in gas sampling vial (Exetainer®, Labco, UK)
and analyzed by a gas chromatograph (Varian 3800, USA) fitted with PoraPLOT Q®
column (Agilent technology, USA) and thermal conductivity detector (TCD). Likewise,
the column temperature was fixed at 100 °C and nitrogen was used as the carrier gas
with a gas flow rate was 3.5 mL/min and the retention time was 4.5 min. Obtained value
was expressed as mmol H2/L media.
Glycerol analysis
During hydrogen production process aqueous samples were collected at regular interval
(24h). Glycerol concentration of such samples were measured by using a
spectrophotometric method of glycerol analysis proposed by (Bondioli et al., 2005).
Briefly (please see the original method for further information), 1 mL of the aqueous
sample (after proper dilution with distilled water) was mixed with 1 mL ethanol (47.5%
v/v). Further, 1.2 mL each of 0.2 M acetylacetone solution and 10 mM sodium periodate
solution (both were prepared by using a 1:1 mixture of 1.6 M acetic acid and 4.0 M
ammonium acetate stock solution) were added to the mixture. Subsequently, the
reaction mixture was incubated at 70 ± 1°C for 1 min by using a rotary shaker water
bath and immediately cooled to 20 ± 1°C by using cold water. The samples were
maintained at the same temperature (by changing the water, if needed) until the optical
density (410 nm) was measured by using an UV-Vis spectrophotometer (Carry 100
Bio®, Varian USA) (Sarma et al., 2013).
The results and discussion portion of the present investigation could be divided into two
parts based on the two different responses considered in the study. In the first part, the
effect of the impurities on hydrogen production has been discussed and the second part
deals with glycerol utilization.
292
Effect of the impurities on cumulative H2 production
The responses (hydrogen production and glycerol utilization) obtained for different runs
of experiments carried out using different concentrations of the impurities have been
presented in Table 4.1.3. Obtained responses of the experiments were analyzed by
using the same software (Design-expert®) used for designing of the experiments.
Response surface linear model was the suggested model for the response observed in
terms of hydrogen production. The ANOVA report for the response surface linear model
has been presented in Table 4.1.4. The model was significant for the observed
response of hydrogen production with insignificant lack of fit and factor C (soap) was the
most significant factor. Similarly, the graphs obtained by analysis of the response for
hydrogen production have been presented in Figure 4.1.2, 4.1.3 & 4.1.4. Figure 4.1.2
represents the response obtained as a function of methanol (factor A) concentration.
Methanol had a strong negative effect on H2 production and the increase in methanol
concentration from 1.3 g/L to 3.3 g/L can result in the decrease in cumulative H2
production. Meanwhile, in an investigation of docosahexaenoic acid production by
bioconversion of crude glycerol, Pyle et al (2008) have mentioned that due to the
increase in methanol concentration from 0 to 20 g/L in the culture, media biomass
productivity could be decreased from 1.92 ± 0.08 g/L-d to 0.94 ± 0.08 g/L-d (Pyle et al.,
2008). Therefore, known inhibitory effect of methanol on microbial growth could be the
most possible reason of present observation.
Likewise, Figure 4.1.3 represents the response of hydrogen production obtained as a
function of NaCl (factor B) concentration. By increasing the NaCl concentration from
0.15 g/L to 0.55 g/L, cumulative H2 production could be improved from around 1.3
mmol/L to 1.6 mmol/L. There may be two reasons for such observation. Firstly, NaCl
might be helping in maintaining the osmotic balance of the system leading to improved
H2 production. Secondly, NaCl may form the salt of organic acids (e.g. sodium acetate
with the acetic acid) produced during the process by which it may improve the buffering
capacity of the system. As pH change due to organic acid production can significantly
inhibit the process, improvement of buffering capacity (due to NaCl) of the media could
enhance the hydrogen production. The present observation is strongly supported by a
293
previous study by (Ngo et al., 2011) where the authors have reported the beneficial
effect of NaCl on H2 production using pre-treated crude glycerol. Thus, it can be
concluded that although NaCl is an impurity in CG, its presence in the concentration
level tested in the present study has no negative effect on the process.
Unlike NaCl, soap (factor C) was found to be inhibitory for hydrogen production.
Moreover, as shown in Table 4.1.4 it is found to be the most significant factor (p value =
0.0104 ˂ 0.05) to influence the process. Figure 4.1.4 is the graphical representation of
the influence of the factor on hydrogen production. As it is clear from the figure, an
increase in soap concentration from 1g/L to 3 g/L can decrease the cumulative
hydrogen production by nearly 33% and reduce the production from around 1.8 mmol/L
to 1.2 mmol/L. Meanwhile, in an investigation of eicosapentaenoic acid production by
bioconversion of biodiesel derived CG Athalye et al (2009) have shown that even a
soap concentration as low as 1 g/L can significantly reduce the biomass as well as the
product yield and it supports our findings (Athalye et al., 2009). Further, possible role of
soap molecules in the disruption of microbial cell membranes could be a reason of our
observation. Thus, in the concentration ranges tested in the present study, methanol
and soap was found to have negative effect on hydrogen production. Therefore, proper
pre-treatment of CG for soap and methanol removal could be the best option for
improved H2 production.
Effect of the impurities on glycerol utilization
Similar to the responses for hydrogen production, glycerol utilization responses
obtained for 20 different runs of experiments have been presented in Table 4.1.3 and
analyzed by Design Expert-7 software. Unlike the data for hydrogen production,
response surface linear model could not be fitted to the data obtained for glycerol
utilization indicating that the factors might have interaction among themselves. All the
graphs generated during the analysis of the data obtained for glycerol utilization have
been shown in Figure 4.1.5. Figure 4.1.5 (a) shows the 3 dimensional response surface
indicating the effect of methanol and NaCl (factor A & B) on glycerol utilization. It could
be determined that the glycerol utilization has been significantly reduced by initial
294
methanol concentration higher than 2.3 g/L. Similar to other alcohols, methanol could be
toxic to bacterial cell either by destroying their cell wall or by altering the functions of
intracellular proteins Whas's toxic and what's not. http://2the4.net/toxic.htm (accessed
on 10/12/2012). Hence, reduced glycerol utilization at higher methanol concentration
could be attributed to detrimental effect of the alcohol to the process organism.
Likewise, maximum glycerol utilization could be observed for initial methanol
concentration of around 2 g/L. Alcohol concentration dependent improvement in cell
permeability (Volpe et al., 2008) and probable improvement in glycerol uptake rate
could be a reason for such response of glycerol utilization. Moreover, at methanol
concentration less than 1.5 g/L slight decrease in glycerol utilization has been observed.
A possible reason for finding of this kind could be that at relatively low methanol
concentration improvement in cell permeability was not sufficient to significantly improve
the substrate utilization; on the contrary, at such concentration methanol may compete
with the substrate for preferential utilization. However, it is a general assumption and it
has to be established by proper investigation. Similarly, from Figure 4.1.5 (a)
corresponding to the increase in NaCl concentration improvement in glycerol utilization
could be observed. Considering NaCl, it is an identical response obtained for both
glycerol utilization and hydrogen production which has been discussed in previous
section. Likewise, a similar response (increase in glycerol utilization) has been observed
(Figure 4.1.5-b) when both soap and NaCl concentration have been simultaneously
increased to maximum. Further, from Figure 4.1.5 (c) it could be summarized that at
constant NaCl (0.3 g/L) and relatively low methanol (around 1.8 g/L) concentration,
percent utilization of glycerol could be slightly increased by increasing the soap
concentration. Meanwhile, surfactant is known to improve the bioavailability of organic
substrate (Sarma et al., 2011); hence, soap mediated improved glycerol utilization could
be the most probable reason of present observation. However, unlike the present
observation (response 2), as already mentioned, a strong negative effect of soap could
be observed for response 1 (hydrogen production) Figure 4.1.4. Thus, it has been noted
that by increasing the soap concentration from 1g/L to 3 g/L glycerol utilization could be
slightly improved however corresponding increase in hydrogen production has not been
295
observed. Metabolic shift, a phenomenon commonly observed in fermentative hydrogen
production (Srikanth et al., 2010) may be a reason for reduced hydrogen production in
presence of relatively higher amount of soap.
Thus, soap and methanol in the initial concentration ranges investigated in the present
study were found to have very strong negative effect on hydrogen production by CG
bioconversion process. Further, Figure 4.1.6 shows the hydrogen production profile
obtained for a control experiment carried out without the addition of any impurity.
Cumulative hydrogen production obtained for the control experiment was higher than all
20 runs of experiment carried out using different concentration of the impurities.
Actually, compared to the control experiment (Figure 4.1.6) 21.56 to 95.31 % reduction
in cumulative H2 production could be observed in the case of 20 runs of experiments
carried out using different concentration of the impurities (Table 4.1.3). This observation
further confirmed the inhibitory effect of the impurities. Meanwhile, the present
investigation was planned considering that 10 g/L CG is optimum for hydrogen
production. Therefore, from the responses obtained from the experimental design
(Table 4.1.3), it could be summarized that hydrogen production could be improved by
diluting the CG below 10 g/L. However, it should also be considered that by diluting the
CG, the glycerol present in CG will also be diluted and it will have a negative effect on
cumulative H2 production of the process. Thus, selective removal of methanol and soap
could be the most obvious option for enhanced CG bioconversion. Methanol could be
evaporated from a CG sample by increasing the temperature beyond 65 °C (Jasuja et
al., 2010, Pyle et al., 2008). Likewise, as it is already described salting out (by adding
NaCl) of soap could be an interesting option. Moreover, NaCl has showed beneficial
response for hydrogen production as well as it might have a role in improving the
buffering capacity (as already discussed good buffering capacity of the media is
beneficial for fermentative H2 production) of the process. Hence, by applying NaCl, a
combined soap removal and pH management strategy could be developed for
enhanced H2 production by CG bioconversion. Presently we are working on this
strategy and it will be communicated as a separate report.
296
Conclusions
Using response surface methodology, role of three different CG impurities (soap,
methanol and NaCl) on hydrogen production and glycerol bioconversion has been
evaluated. Among these impurities, soap (1 to 3 g/L) was found to have significant (p
value = 0.0104 ˂ 0.05) inhibitory effect on hydrogen production. Likewise, methanol (1.3
to 3.3 g/L) has also showed similar response in the concentration range considered.
Interestingly, for both the impurities, the response for glycerol utilization was not similar
to the response obtained hydrogen production (as expected). Co-utilization of the
impurities along with glycerol as well as a metabolic shift due to the presence of the
impurities was identified as most probable reason for such observation. Meanwhile,
NaCl was found to have beneficial effect for both H2 production and glycerol utilization.
Based on the observations a novel strategy involving NaCl has been proposed for
combined soap removal and buffer capacity improvement of CG based media.
Acknowledgments
Authors would like to thank Le Centre de Recherche Industrielle du Québec (CRIQ),
MAPAQ (No. 809051), Natural Sciences and Engineering Research Council of Canada
(Discovery Grant 355254) and Ministère des Relations Internationales du Québec
(coopération Paraná-Québec 2010-2012) for financial support.
297
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300
Table 4.1. 1: Distribution of different impurities of biodiesel derived crude glycerol
CG impurity
Presence in CG
Minimum
conc.
of
the impurity present
Minimum conc. present in 10
Reference
g/L CG
in CG
Methanol
23.4-37.5 % (w/w)
23%w/w
2.3 g/L
(Tang et al., 2009, Thompson et al., 2006)
Sodium
3.12 % (w/w)
3.5% w/w
0.35 g/L
http://rendermagazine.com/articles/2008-
chloride
issues/2008-august/2008-08-crude-glycerin/
(accessed on 03_09_2012)
Soap
20.5-31.4 % (w/w)
20 % w/w
2.0 g/L
301
(Hu et al., 2012)
Table 4.1. 2: Central composite design ranges of the three factors considered for present investigation.
Sl. no.
Variables
Symbol
Variable concentration (coded level) g/L
-α
Low
Mid
High
+α
1
Methanol
A
0.62
1.30
2.3
3.3
3.98
2
Sodium
B
0.01
0.15
0.35
0.55
0.69
C
0.32
1.00
2.0
3.00
3.68
chloride
3
Soap
302
Table 4.1. 3: Experimental design and the responses obtained for 20 different experiments carried out during the present study
Run
Independent variables
Methanol (g/L)
NaCl (g/L)
Responses
Soap (g/L)
H2 (mmol/L)
1
2.30
0.35
2.00
1.68
78.57
2
3.30
0.55
3.00
1.75
81.42
3
3.30
0.15
3.00
1.11
68.57
4
2.30
0.35
0.32
2.51
80
5
2.30
0.35
2.00
1.34
65.71
6
2.30
0.35
2.00
1.75
68.57
7
0.62
0.35
2.00
1.27
58.57
8
1.30
0.55
1.00
1.96
55.71
9
3.30
0.55
1.00
2.01
65.71
10
2.30
0.35
2.00
1.84
64.28
11
2.30
0.69
2.00
1.68
80
303
Run
Independent variables
Methanol (g/L)
NaCl (g/L)
Responses
Soap (g/L)
H2 (mmol/L)
12
1.30
0.55
3.00
1.28
72.85
13
1.30
0.15
3.00
1.28
77.14
14
3.30
0.15
1.00
1.65
78.57
15
2.30
0.35
3.68
1.09
78.57
16
1.30
0.15
1.00
1.81
74.28
17
2.30
0.35
2.00
1.09
71.42
18
2.30
0.01
2.00
0.9
51.42
19
2.30
0.35
2.00
1.4
60
20
3.98
0.35
2.00
0.15
18.57
304
Table 4.1. 4: ANOVA for response surface linear model used for response 1 (hydrogen production)
Source
Sum of squares
Degree of freedom
Mean square
F value
P value
Model
2.07
3
0.69
4.10
0.0245*
A-Methanol g/L
0.21
1
0.21
1.25
0.2803
B-NaCl g/L
0.44
1
0.44
2.64
0.1239
C-Soap g/L
1.42
1
1.42
8.42
0.0104*
Residual
2.69
16
0.17
-
-
Lack of Fit
2.28
11
0.21
2.51
0.1598**
Cor Total
4.76
19
-
-
-
305
Figure 4.1. 1: Schematic representation of soap separation from crude glycerol sample
Figure 4.1. 2: Response surface graph showing the effect of methanol on hydrogen production
308
Figure 4.1. 3: Response surface graph showing the effect of NaCl on hydrogen production
309
Figure 4.1. 4: Response surface graph showing the effect of soap on hydrogen production
310
311
312
Figure 4.1. 5: Response surface graphs showing the effect of (a) methanol and NaCl; (b) NaCl and
soap and (c) soap and methanol on glycerol utilization
313
Figure 4.1. 6: Hydrogen production profile obtained for the control experiment carried out in the
absence of any impurity
314
PART II
MITIGATION OF THE INHIBITORY EFFECT OF SOAP BY MAGNESIUM
SALT TREATMENT OF CRUDE GLYCEROL- A NOVEL APPROACH
FOR ENHANCED BIOHYDROGEN PRODUCTION FROM THE
BIODIESEL INDUSTRY WASTE
Carlos Ricardo Soccolc
a
Institut National de la Recherche Scientifique, Centre - Eau Terre Environnement, 490,
Rue de la Couronne, Québec(QC), Canada G1K 9A9
b
c
[email protected])
Bioresource Technology (2014) 151: 49-53.
315
Résumé
En raison de son effet inhibiteur sur la croissance microbienne, le savon présent dans le
glycérol brut (GB) est un sujet de préoccupation pour la valorisation biologique des
déchets de fabrication du biodiesel. Par une stratégie de relargage (Salting out), jusqu'à
42% du savon a été retiré du milieu et l'approche a un effet bénéfique sur la production
de H2. Toutefois, les expérimentations montrent que l'élimination de plus de 7% du
savon a été défavorable à la production de H2. En fait, le savon, sous certaines
concentrations, est utilisé comme co-substrat et son élimination affecterait le rapport
carbone-azote du milieu diminuant ainsi la production de H2. Sans modifier le rapport
carbone-azote du GB, le traitement au MgSO4 permet de convertir le savon en sa forme
inactive (écume). L'approche a permis d’augmenter le taux de production de H2 de
33,8%, la production cumulative de H2 de 34,7% et la consommation du glycérol (près
de 2,5 fois). Également, le traitement permet d’augmenter le Mg (un nutriment) contenu
dans le milieu de culture support de 0,57 mg/L à 202mg/L.
Mots clés: biodiesel; biohydrogène; glycérol brut; impureté; savon; magnésium
316
Abstract
Owing to its inhibitory effect on microbial growth, soap present in crude glycerol (CG) is
a concern in biological valorization of the biodiesel manufacturing waste. By salting out
strategy, up to 42% of the soap has been removed and the approach has beneficial
effect on H2 production; however, removal of more than 7% of the soap was found to be
inhibitory. Actually, soap is utilized as a co-substrate and due to removal; the carbonnitrogen ratio of the medium might have decreased to reduce the production.
Alternatively, without changing the carbon-nitrogen ratio of CG, MgSO4 treatment can
convert the soap to its inactive form (scum). The approach was found to increase the H2
production rate (33.82%), cumulative H2 production (34.70%) as well as glycerol
utilization (nearly 2.5 folds). Additionally, the treatment can increase the Mg (a nutrient)
content of the medium from 0,57 mg/L to 202 mg/L.
Key words: Biodiesel; biohydrogen; crude glycerol; impurity; soap
317
Introduction
At present, global biodiesel production is around 30 billion liter and it is projected to
reach 42 billion liter mark by 2021 (www.fao.org, 2013). During biodiesel production by
transesterification of lipids (e.g. vegetable oils, animal fats and algal lipids, among
others), crude glycerol (CG) is generated as a byproduct; and by weight, it is around
10% of the biodiesel produced (Johnson et al., 2007). Due to presence of numbers of
impurities as well as continuous increase in its global production (corresponding to the
increase in biodiesel production), the present market value of CG is very low. According
to a report by Yang et al. (2012), the price of CG is only around $ 0.05/pound. Likewise,
due to high pH, high COD/BOD, presence of methanol as well as its viscous nature,
environmental disposal of CG is a challenge for biodiesel manufacturers (Dobson et al.,
2012). Alternatively, CG can be used as a feedstock for microbial hydrogen production
and in the context of global interest for the development of renewable energy sources, it
can be considered as a promising technology.
As already mentioned, CG contains a range of impurities and soap is one of them (Hu
et al., 2012; Pyle et al., 2008). According to Hu et al. (2012), soap content of different
CG sample may vary from 20.5 ± 0.1 to 31.4 ± 0.1 % (w/w). Unfortunately, soap is a
potential inhibitor of microbial growth (Athalye et al., 2009). Therefore, removal of the
soap from CG may have beneficial effect on microbial growth and enhanced hydrogen
production can be expected by CG bioconversion. Thus, in the present study NaCl and
MgSO4 based treatment strategies to mitigate the inhibitory effect of soap present in CG
has been demonstrated. Enhancement in hydrogen production and glycerol utilization
has been determined to evaluate the approaches. Additionally, potential beneficial effect
of soap present in CG on hydrogen production has also been explored. Enterobacter
aerogenes, a well-known hydrogen producer (Fabiano and Perego, 2002) has been
used in the present study.
318
Chemicals and reagents
Biodiesel manufacturing waste (crude glycerol) was supplied by Rothsay, Canada and
after proper dilution (as specified below) it was used as the substrate for hydrogen
production. Glucose, NaCl, MgSO4, NaOH, HCl, acetone, ethanol, bromophenol blue,
sodium periodate, acetylacetone, ammonium acetate and acetic acid were purchased
from Fisher scientific (Mississauga, Ontario, Canada). Yeast extract was provided by
Lallemand (Montreal, Canada) and casein peptone and KH2PO4 was purchased from
VWR (Mississauga, Ontario, Canada).
Microorganism and culture conditions
Enterobacter aerogenes NRRL B 407, a facultative anaerobic bacterium provided by
ARS, USDA, USA, was used for all the hydrogen production experiments conducted in
this study. The microorganism was maintained by regular sub-culturing using a nutrient
medium containing glucose (5 g/L), casein peptone (5 g/L), KH2PO4 (2 g/L) and yeast
extract and MgSO4 (0.5 g/L each). For this purpose, 125 mL serum bottles were used
and in each bottle, about 50 mL of the medium was taken. The serum bottles containing
the media were converted to anaerobic bioreactor by passing ultrapure N2 gas (for 2
min) through the media and they were immediately sealed with aluminum crimp seal
containing silicone septum. A hand operated crimper (E-Z Crimper™, VWR, Canada)
was used for sealing the bottles. After autoclaving, the bottles were cooled to room
temperature and they were inoculated (using sterile syringe fitted with fine needle) with
5 % (v/v) microbial suspension from preceding culture. Finally, the bottles were
incubated in an incubator shaker operated at 150 rpm and 30°C. A bacterial cell
suspension obtained by the above procedure with the microorganisms in their
exponential phase of growth was used as inoculum (5% v/v) for all hydrogen production
experiments conducted during the present investigation (Sarma et al., 2013).
319
CG pretreatment using NaCl
A crude glycerol sample with initial pH of 11.37 has been considered for this
investigation. Due to high viscosity, the original sample was diluted with distilled water
to prepare a CG solution of 200 g/L. To determine the effect of NaCl concentration on
soap removal from crude glycerol, 50 mL of 200 g/L CG was separately mixed with
different concentrations of NaCl (1, 5, 10, 20, 50, 100, 200 and 300 g/L) by using a
magnetic stirrer. After complete solubilization of NaCl, precipitated soaps were removed
by centrifuging the mixtures at 5000 g for 20 min. Soap concentration of untreated and
NaCl treated CG samples were determined (as described later) and soap removal
efficiency of different NaCl treatment was expressed as percent soap removal. NaCl
containing supernatants were properly diluted (as specified in next sections) with
distilled water to get required concentration of CG and used as the feedstock for
hydrogen production.
Bio-hydrogen production using NaCl treated CG
Optimum CG concentration for biohydrogen production by E. aerogenes NRRL B 407
was found to be 10 g/L (Sarma et al., 2013). Therefore, to determine the effect of NaCl
treatment, CG treated with different concentrations of NaCl (viz. 1, 5, 10, 20, 50, 100
and 200 g/L of NaCl) were diluted with distilled water to obtain a final CG concentration
of 10 g/L. For each experimental run, 25 mL of NaCl treated diluted CG (i.e. 10 g/L) was
taken in 125 mL serum bottle (VWR, Canada) and the medium pH was adjusted to 6.0
(which is optimum for hydrogen production by E. aerogenes (Marques et al., 2009)). An
experiment was also conducted using untreated CG of similar concentration and it was
considered as negative control. As already mentioned, to achieve anaerobic condition,
oxygen was driven out of the medium by passing ultrapure N2 gas using a fine needle
fitted silicon tube and the bottle was immediately sealed. Sealed bottles were sterilized
(by autoclaving) and after cooling to room temperature, they were inoculated with 5 %
(v/v) fresh microbial suspension. Inoculum preparation, inoculation technique and other
culture conditions were same as mentioned above. During incubation period (120h), at
12/24h interval, 1 mL of the produced gas was collected from the headspace of each
320
serum bottle by using a 5 mL gastight syringe (SGE Analytical Science Pvt. Ltd,
Australia). Likewise, 1 mL of aqueous sample was collected at 24 h interval by using a
sterile syringe. Collected gas and liquid samples were analyzed by standard analytical
techniques which are separately described.
Bio-hydrogen production using MgSO4 treated CG
For this experiment, 20 g/L of CG was used as the substrate for hydrogen production
and four different concentrations of MgSO4 (viz. 0.5, 1, 2.5 and 5 g/L) have been tested.
Specified amount of MgSO4 was separately mixed with 25 mL of CG taken in different
serum bottles (125 mL) and to evaluate the effect of MgSO4 treatment on hydrogen
production, the bottles were further prepared, inoculated and incubated in the same
manner as described for NaCl treated CG.
Analytical methods
Determination of soap concentration in CG samples
To determine the amount of soap present in CG, a titrimetric method was used and the
concentration was expressed as sodium oleate equivalent (Sarma et al., 2013). Briefly,
150 µL of 0.5% (w/v) bromophenol blue prepared in 95% ethanol was mixed with 60 mL
of acetone. Further, the solution was neutralized with 0.1 M NaOH and was mixed with
10 g of CG sample (NaCl treated and untreated). Subsequently, for 1 min the solution
was placed in a water bath maintained at 70°C and after cooling to room temperature, it
was titrated using 0.1 M HCl. Disappearance of blue color of the solution was an
indication of completion of the titration and the amount of the acid consumed was used
for calculating the soap content of different CG samples (Sarma et al., 2013).
Hydrogen analysis
To determine the concentration of hydrogen accumulated in the headspace of the
serum bottles (used for hydrogen production), the samples were analyzed by gas
chromatography. As already mentioned, 1 mL of gaseous sample was collected from
each bottle, at 12/24h interval for 120h by using gastight syringe and they were kept in
5.9 mL gas sampling vial (Exetainer®, Labco, UK). The gas chromatographic analysis
321
was performed by using a Varian 3800 gas chromatograph (Varian, USA) and the
column and detector used for the analysis were PoraPLOT Q® (Agilent technology,
USA) and thermal conductivity detector (TCD), respectively. The carrier gas used for
this analysis was nitrogen, the flow rate was 3.5 mL/min and the column temperature
was 100 °C (Sarma et al., 2013).
Glycerol analysis
The liquid samples collected during hydrogen production were analyzed for residual
glycerol concentrations by using the same method as already described in (Sarma et
al., 2013; Bondioli and Della Bella, 2005). Briefly, the original sample was diluted with
distilled water and 1 mL of the same was mixed with 1 mL of ethanol (47.5 % v/v). The
resulting solution was mixed with 1.2 mL of acetylacetone solution (0.2 M) and 1.2 mL
of sodium periodate solution (10 mM). Further, the reaction mixture was placed in a
water bath maintained at 70°C (for 1 min) and subsequently transferred to another
water reservoir containing cold water (20°C). Finally, the samples were analyzed (410
nm) by an UV-Vis spectrophotometer (Varian, USA) (Sarma et al., 2013).
NaCl pretreatment and bio-hydrogen production using treated CG
Addition of salt (NaCl) can increase the solubility of glycerol (glycerol is more soluble in
saline water) in a crude glycerol sample and therefore, the soap present in the solution
can be precipitated and removed by centrifugation (PCE, 2013). Due to high viscosity,
the original sample of CG was not suitable for centrifugal removal of precipitated soap.
Therefore, it was first diluted with distilled water to prepare a CG solution of 200 g/L and
then treated with different concentrations of NaCl. Eight different concentrations of NaCl
(1, 5, 10, 20, 50, 100, 200 and 300 g/L) were evaluated for soap removal. The
concentration, 300 g/L is almost close to the solubility limit of NaCl; hence, more than
300 g/L was not considered. 1 g/L NaCl was unable to remove any soap; whereas, 42
% soap removal was achieved for 300 g/L of NaCl. Further, crude glycerol treated with
different concentrations of NaCl was evaluated as the feedstock for hydrogen
production. Figure 4.2.1 shows cumulative hydrogen production obtained (after 120h of
322
incubation) by bioconversion of 10 g/L of crude glycerol treated with different
concentrations of NaCl. From Figure 4.2.1, it can be concluded that treatment of CG by
5, 10 or 20 g/L of NaCl has beneficial effect on cumulative hydrogen production; where,
a maximum 19.28 % improvement in hydrogen production has been observed for CG
treated with 10 g/L NaCl (corresponding to 5% soap removal). However, further
increase in NaCl concentration can drastically inhibit the production; where, maximum
93.03 % decrease in hydrogen production (Figure 4.2.1) has been observed for CG
treated with 200 g/L NaCl (corresponding to 31% soap removal). One probable reason
for such observation can be that 9-31 % removal of soap (corresponding to 50-200 g/L
NaCl treatment) might decrease the C-N (carbon-nitrogen) ratio of CG based media to
an inhibitory level. It is well known that slight change in C-N ratio can significantly affect
the hydrogen production process (Argun et al., 2008; Lin and Lay, 2004). This
observation also indicates that although, soap is largely considered as an impurity of
CG; still it has a role in hydrogen production and most possibly, along with glycerol it is
used as a co-substrate by the bacterium.
It should be noted that during NaCl treatment of CG, NaCl was completely dissolved in
the CG solution. Therefore, apart from CG, the hydrogen production media (prepared by
diluting the CG treated with different concentrations of NaCl) also contained different
concentrations of NaCl (Figure 4.2.1). Hence, presence of relatively high concentration
of NaCl in the media might be a reason for reduced hydrogen production in the case of
50-200 g/L NaCl treated CG. To verify this possibility, an experiment was conducted
where 200 g/L NaCl treated CG was diluted with distilled water to an extent so that it
contained only 0.5 g/L NaCl in the final media (as maximum hydrogen production was
obtained for 10 g/L NaCl treated CG where NaCl concentration in the final medium was
0.5 g/L -Figure 4.2.1). As shown in Figure 4.2.2, the results clearly indicate that
irrespective of NaCl concentration in the medium (within the concentration range tested
in the present investigation) separation of the soap from CG has negative effect on
hydrogen production. Thus, as already mentioned non-availability of soap as a cosubstrate and change in C-N ratio of the media due to separation of the soap from CG,
can be the main reasons of observed reduced hydrogen production.
323
Final pH of the media left after hydrogen production by bioconversion of CG treated with
different concentrations of NaCl can vary from 3.96 ± 0.03 to 3.75 ± 0.03. It has been
observed that corresponding to an increase in soap removal due to NaCl treatment,
there was a decrease in final pH of the media. The result indicates that soap might have
played a role of buffering/acid neutralizing agent in CG based media and it has the
capacity to minimize the pH drop due to organic acid production during bio-hydrogen
production.
Biohydrogen production using MgSO4 treated CG
From aforementioned findings, it is clear that although soap is considered as an impurity
of CG and it has known inhibitory effect on microorganism; yet, presence of soap in CG
is beneficial for fermentative hydrogen production. Therefore, if without separating it
from CG, the functional property of the soap molecule could be destroyed (so that its
inhibitory effect could be mitigated); enhanced H2 production by CG bioconversion may
be possible. It is well known that hard water contains Mg2+ or Ca2+ ions which react with
soap (www.mrwa.com, 2013). Mg2+ ion binds with soap molecules to form scum, which
is an ineffective form of soap. Therefore, MgSO4 was added to the CG based medium
so that soap molecules present in the solution could be converted to scum and at the
same time they remain as a part of the solution (unlike salting out). Figure 4.2.3a &
4.2.3b show the hydrogen production and glycerol utilization profiles obtained by
bioconversion of CG treated with four different concentrations of MgSO4 (0.5 g/L, 1 g/L,
2.5 g/L and 5 g/L). It is clear that 0.5 g/L MgSO4 was unable to show any significant
improvement in hydrogen production. In the case of 1 g/L MgSO4 treated CG, during
active production phase, the hydrogen production rate was 33.82% higher than that of
untreated CG. Similarly, cumulative hydrogen production was found to be enhanced by
34.70%. Thus, the approach has the potential to significantly reduce the duration of the
fermentation with simultaneous increase in cumulative hydrogen production. Apart from
its role as a “killer of soap”, MgSO4 might have played a role of additional nutrient for
the bacterium. This assumption is further supported by the fact that Mg is a part of the
culture media used for Enterobacter (Zhen-qian and Chun-yun, 2009) and crude
glycerol contains only 57.41 ± 18 ppm of the same (Sarma et al., 2013). For hydrogen
324
production, CG has to be diluted to a fermentable concentration (e.g. 10 g/L) and during
this process Mg concentration is further decreased to around 0.57 ppm. Moreover, this
small concentration of Mg naturally present in CG can react with soap to form scum,
and become unavailable for microbial utilization. Thus, application of MgSO4 has dual
benefits for the process. However, further increase in MgSO4 concentration was not
found to have any beneficial effect, in fact CG treated with 5 g/L MgSO4 was found to
inhibit the hydrogen production by 75.94%. The glycerol utilization profiles presented in
Figure 4.2.3b shows that compared to 8.65% glycerol utilization in the case of untreated
CG (control) nearly 2.5 folds increase (around 20.94%) in glycerol utilization has been
observed in the case of 1 g/L MgSO4 treated CG. Similarly, in the case of 0.5 g/L, 1 g/L
and 2.5 g/L MgSO4 treated CG, the rates of glycerol utilization were found to be higher
than that of untreated CG. A possible explanation of improved glycerol utilization is that
due to MgSO4 treatment, soap scum was formed and a portion of the soap molecules
became temporarily unavailable (as co-substrate) for the bacterium; hence, more
glycerol was utilized. This observation further ascertains the assumption that although
soap is commonly considered as an impurity of CG (Pyle et al., 2008; Athalye et al.,
2009; Ethier et al., 2011); during hydrogen production along with glycerol it is also
utilized as a co-substrate.
The purpose of the present investigation was to determine the effect of NaCl and
MgSO4 treatment on hydrogen production by CG bioconversion. Both the treatments
have shown beneficial effect on the process. Especially, owing to negligible Mg content
of CG, application of magnesium salt seems to be indispensable for improved CG
bioconversion. Similarly, for the first time, the evidence of beneficial role of soap
(present in CG) on CG bioconversion has been obtained. Thus, the present
investigation has covered an unexplored side of CG bioconversion process. However,
these are only preliminary findings and to be suitable for industrial application, the
process needs further improvement in a numbers of aspects. Firstly, the concentration
of MgSO4 and CG should be optimized in such a way, so that the requirement of MgSO 4
can be reduced and complete utilization of CG can be achieved. Secondly, the closed
batch mode of fermentation used for the present investigation is suitable only for the
325
study of the effect of different treatments. However, hydrogen accumulation in the
headspace of the reactor is inhibitory for the process (van Niel et al., 2003). Hence,
continuous separation of produced gas should be considered for large scale hydrogen
production. Similarly, in the present investigation, sterilized CG was used as the
feedstock. However, considering the cost and energy requirement, feedstock
sterilization may not be a feasible option for large scale hydrogen production. Compared
to conventional feedstock such as, wastewater/wastewater sludge, CG is expected to
have a lesser load of indigenous microorganism. Therefore, in the case of hydrogen
production by CG bioconversion, presence of potential hydrogen consumers such as,
methanogens is not expected. Thus, it will be interesting to study the hydrogen
production potential of unsterile CG.
Conclusions
Salting out can remove around 42% of the soap present in a CG; however, along with
glycerol it is utilized as a co-substrate and its removal may inhibit the H2 production by
decreasing the C-N ratio of the medium. Alternatively, without removing it from the
medium, MgSO4 treatment of CG can convert the soap to its inactive form (scum). The
novel approach can increase the cumulative H2 production by 34.70% and glycerol
utilization by nearly 2.5 folds. Additionally, as CG contains only 57.41 ± 18 ppm of Mg,
MgSO4 addition can also improve the nutritional quality of the feedstock.
Acknowledgments
Authors are thankful to Le Centre de Recherche Industrielle du Québec (CRIQ) and
NSERC for financial support. Fonds Québécois de la Recherche sur la Nature et les
Technologies (FQRNT) has also been acknowledged for financial assistance through a
doctoral research fellowship.
326
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fermentation of wheat powder solution: effects of C/N and C/P ratio on hydrogen
yield and formation rate. International Journal of Hydrogen Energy 33 (7): 18131819.
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determination of free glycerol in biodiesel. European Journal of Lipid Science and
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Dobson R, Gray V & Rumbold K (2012) Microbial utilization of crude glycerol for the
production of value-added products. Journal of Industrial Microbiology and
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from glycerol by a strain of Enrerobacter aerogenes. In: proceedings of the
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329
40
H2 production (mmol/L)
12
Hydrogen
NaCl
10
35
30
8
25
6
20
15
4
10
2
5
0
NaCl conc. in the media (g/L)
45
0
1g/L
5g/L
10g/L
20g/L
50g/L 100g/L
NaCl used for salting out
200g/L Control
Figure 4.2. 1: Cumulative H2 production (after 120h) obtained using 10 g/L CG (treated with
different concentration of NaCl). Initial NaCl concentration in the medium of each run of
experiment was also shown
330
3.5
3
2.5
2
1.5
1
0.5
0
After soap rewmoval
Without soap removal
Figure 4.2. 2: Cumulative H2 production obtained using 200 g/L NaCl treated CG diluted to an
initial NaCl concentration of 0.5 g/L (as from Figure 1, 0.5 g/L NaCl was found to be optimum).
Control represents result obtained for equivalent amount of untreated CG
331
0.5 g/L
1 g/L
2.5 g/L
5 g/L
30
25
20
15
10
5
0
0
10
20
30
Time (h)
3a
332
40
50
60
0.5 g/L
1 g/L
2.5 g/L
5 g/L
Control
105
Residual glycerol (%)
100
95
90
85
80
75
0
10
20
30
40
50
60
Time (h)
3b
Figure 4.2. 3: H2 production (a) and glycerol utilization (b) profiles obtained for 20 g/L CG treated
with different concentrations of MgSO4. Control represents result obtained for untreated CG
333
CHAPTER 5
APPLICATION OF NANOTECHNOLOGY FOR IMPROVED CRUDE
GLYCEROL BIOCONVERSION
Chapter 5. Application of nanotechnology for improved crude glycerol bioconversion
ENRICHED HYDROGEN PRODUCTION BY BIOCONVERSION OF
BIODIESEL WASTE SUPPLEMENTED WITH FERRIC CITRATE AND
ITS NANO-SPRAY DRIED PARTICLES
Saurabh Jyoti Sarmaa, Satinder Kaur Brara, Jérémy Reignerb, Yann Le Bihanc,
Gerardo Buelnac
a
b
Université Paris-Est Créteil Val de Marne, 61 avenue du Général de Gaulle, 94010
Créteil Cedex, France
c
[email protected])
RSC Advances (2014) 4 : 49588-49594.
337
Résumé
L’augmentation de la consommation des combustibles fossiles non renouvelables ainsi
que l'inquiétude suscitée par la pollution et les changements climatiques ont accéléré le
développement de l'industrie des biocarburants durables. Le biodiesel, le bioéthanol et
le biométhane sont disponibles au niveau commercial comme des alternatives des
combustibles fossiles. Actuellement, des recherches visent à trouver un biocarburant
plus respectueux de l'environnement, de préférence produit à partir de matières
premières non alimentaires et capable de satisfaire aux exigences énergétiques des
transports. Dans ce contexte, le biohydrogène produit à partir des déchets organiques
produit de l'eau comme seule émission importante lors de la combustion. De plus,
l’hydrogène est un candidat idéal en raison de son contenu énergétique massique plus
élevé par rapport aux carburants fossiles et biocarburants actuels. Dans la présente
recherche, le glycérol brut (GB) comme sous-produit de production de biodiesel a été
utilisé comme matière première pour la production de biohydrogène et différents
suppléments ont été testés pour augmenter le rendement de la fermentation. Le citrate
ferrique et ses nanoparticules produites par atomisation à sec ont permis d’améliorer la
production d'hydrogène par 50,5 %. La production d'hydrogène utilisant des
concentrations extrêmement faibles en GB de 100 mg/L a permis de produire 22,7 molH2/kg GB ce qui est supérieur à 2,75 fois la valeur de 8,3 mol-H2/kg GB répertoriée pour
la fermentation sombre.
Mots clés: biohydrogène; biodiesel; glycérol brut; Le citrate ferrique; nanoparticule
338
Abstract
Increasing consumption of non-renewable fossil fuels as well as the concern over
pollution and global climate change has accelerated the development of sustainable
biofuel industry. Biodiesel, bioethanol and biomethane are already commercially
available as alternatives of fossil fuels and the search for a more environmental friendly
biofuel, preferentially produced from non-food raw materials and capable of fulfilling the
transportation energy requirement of the globe for longer duration, is going on. In this
context, biohydrogen produced from waste biomass; which produces water as the only
major emission during combustion and has higher energy content compared to fossil
and biofuels of equivalent mass, is an ideal candidate. In the present investigation,
crude glycerol (CG) generated as a by-product of biodiesel production process has
been used as a feedstock for biohydrogen production and different supplements have
been tested for increasing the product yield. Ferric citrate and its nano-spray dried
particles have been found to enhance the hydrogen production by 50.45 %. Hydrogen
production using extremely low CG concentration of 100 mg/L has been found to
produce 22.7 mol-H2/kg CG; which is 2.75 folds higher than 8.25 mol-H2/kg CG known
for dark fermentation.
Key words: Biohydrogen; Biodiesel; Crude glycerol; Ferric citrate; Nanospray dried
particles
339
Introduction
Considering global energy security as well as greenhouse gas emission reduction
potential, increased production and application of biofuels is inevitable. At this juncture,
biofuel production is not equally profitable in all countries and hence, different
governments have applied encouraging policies, such as tax exemption (Banse et al.,
2013). Moreover, in order to ensure increased biofuel consumption and replacement of
fossil fuel, mandatory blending of fossil fuels with 2-10 % of biofuel has been targeted in
many countries (Banse et al., 2013). There are different types of biofuels presently in
use; however, greenhouse gas emission reduction potential, availability of raw materials
and energy content of these biofuels are not similar. Therefore, for long term production
and use, all of them may not be equally suitable (Hay et al., 2013). In this context,
biohydrogen could be considered as an emerging biofuel with very high sustainability. It
can be produced from a diverse group of renewable raw materials and hence, does not
necessarily compete with food crop based biomass for feedstock. Additionally, during
combustion, hydrogen is converted to water and does not produce any carbon based
emission (Hay et al., 2013, Mohd Yasin et al., 2013, Sydney et al.). Energy content of
hydrogen is 142 kJ /g, which is higher than any fossil fuel of equivalent mass (Hay et al.,
2013, Sydney et al.). Thus, hydrogen production by bioconversion of low cost agroindustrial organic waste materials has commercial importance. In the present study,
enhanced hydrogen production by bioconversion of crude glycerol has been
investigated. In the course of biodiesel production by trans-esterification of vegetable
oil, animal fat or any other lipid based feedstock; crude glycerol (CG) is produced as a
by-product. By weight, the amount of glycerol generated by the process could be as
high as 10 % of the biodiesel produced (Yang et al., 2012). Corresponding to increased
global biodiesel production, crude glycerol production is also increasing and
consequently, CG price has decreased to around $ 0.05/pounds (Yang et al., 2012).
Thus, application of CG as a feedstock for hydrogen production by anaerobic
fermentation could be considered as a lucrative option for its value-addition.
340
[NiFe]-hydrogenase and [FeFe]-hydrogenase are the key enzymes responsible for
fermentative hydrogen production (Schmidt et al., 2011). Ni and/or Fe cofactors are
indispensable part of the active sites of these enzymes (Volbeda et al., 1996).
Therefore, it has been postulated that sufficient amount of Ni and Fe should be present
in the fermentation media for improved hydrogen production. Based on this assumption,
in the present study, fermentation media prepared using CG have been supplemented
with ferric citrate, FeSO4, nickel acetate, and NiCl2 and their effects on hydrogen
production have been investigated. Owing to their very large specific surface area,
nanoparticles are generally considered to have higher reactivity (Van Leeuwen et al.,
2013). Therefore, aforementioned supplements were used in nanospray dried form. For
the present investigation, Enterobacter aerogenes, a well-known hydrogen producing
microorganism has been used (Tanisho, 1998, Zhang et al., 2011). CG used in this
investigation was supplied by Rothsay®(Canada) and its detailed characterization could
be found in Sarma et al. (2013).
Materials and Methods
Enterobacter aerogenes NRRL B-407, a rod-shaped, gram-negative, facultative
anaerobic bacterium from Enterobacteriaceae family has been used for hydrogen
production experiments. It was collected from ARS, USDA, USA in freeze-dried form.
To subculture the organism and to prepare inoculum for the study, a synthetic medium
composed of glucose and casein peptone (5 g/L each); KH2PO4 (2 g/L); and yeast
extract and MgSO4.7H2O (0.5 g/L each) was used. Serum bottles of 125 mL with 50 mL
of working volume were used to culture the microorganism. Media taken in these bottles
were first sparged with N2 gas for 2 min; the bottles were immediately closed with
aluminum crimp seals having silicon septa and sterilized by autoclaving. After
inoculation, the culture bottles were incubated in an incubator shaker operated at 30°C
and 150 rpm and 5 % (v/v) of microbial culture at its exponential phase of growth was
used as an inoculum for hydrogen production.
341
Preparation and characterization of different nano-spray dried particles
Ferric citrate, FeSO4, nickel acetate, and NiCl2 particles used in the present
investigation have been prepared by using a nano-spray dryer (Buchi B-90,
Switzerland). For the preparation of FeSO4, nickel acetate, and NiCl2 particles, a
solution of 50 g/L of each compound was separately prepared in distilled water and
subjected to nano-spray drying process. For all particles, the respective solution was
fed to the nano-spray dryer at a flow rate of nearly 20 mL/h (flow level 3). Similarly, in
each case, the maximum temperature of the spray dryer was kept constant at 120 °C
and the air flow rate was maintained at 120L/min. The spray cap used for entire spray
drying process was of 4.0 μm mesh size. During the process, particles were
accumulated on the internal surface of the collecting electrode which was subsequently
recovered by particle collecting accessories supplied with the instrument. Collected
nano-spray dried particles were used in hydrogen production experiments. For
preserving the particles, airtight glass vials were used. For the preparation of ferric
citrate particles, almost similar process was applied. However, as ferric citrate solution
could not be prepared using water in room temperature; either hot water or 1N NaOH
was used.
Zeta potential distribution of nano-spray dried ferric citrate particles prepared using hot
water was determined by a zetasizer nano ZS system (Malvern instruments Ltd., UK).
For this purpose, small amount of the particles was dispersed in Milli-Q water and 1 mL
of this solution was analyzed.
A scanning electron microscope (Zeiss EVO® 50 Smart SEM) has been used for
morphological characterization of the nano-spray dried particles prepared for this
investigation. The ferric citrate nano-spray dried particles were first dispersed in Milli-Q
water; a small amount of dispersed nano-spray dried particles was taken on a piece of
aluminum foil, air dried and finally transferred to SEM stub. Prior to SEM analysis,
samples were coated with gold by using a sputter coater.
342
Screening of different nano-spray dried particles for biohydrogen production
Purpose of this investigation was to investigate the effect of nano-spray dried particles
prepared using ferric citrate, FeSO4, nickel acetate, and NiCl2, on hydrogen production
by CG bioconversion. All the particle types were prepared using aqueous solution as
already mentioned in previous section (2.2). CG stock solution was distributed in
different 125 mL serum bottles in such a way that after inoculating with 5 % (v/v)
inoculum, total volume of the medium and concentration of CG in each bottle would
become 25 mL and 12 g/L, respectively. About 50 mg/L of each nano-spray dried
particle type was separately added to different bottles and the medium pH of each bottle
was adjusted to 6. Nitrogen was passed through the media; bottles were sealed,
sterilized, inoculated, and incubated in the same manner as described in section 2.1.
One mL of biogas containing hydrogen was collected from the headspace of each bottle
using a 5 mL gastight glass syringe (SEG, Australia) and stored in 5.9 mL airtight glass
vials (Exetainer®, Labco, UK). Prior to storing the gas samples, glass vials were flashed
with ultrapure nitrogen gas and evacuated using a needle fitted vacuum pump.
Investigation of the effect of Fe-citrate particle preparation method on hydrogen
production
As mentioned in section 2.2, ferric citrate was dissolved either in hot water or in NaOH
(1N); which gave two different types of solutions. From these two solutions, two different
types of ferric citrate particles were prepared by nano-spray drying process. About 10,
50, 100, 300 and 500 mg/L of both particles were separately added to different serum
bottles containing 12 g/L CG and their effect on hydrogen production was investigated
by similar method mentioned in section 2.3.
Investigation of the effect of CG concentration on H2 production using media
supplemented with ferric citrate particles
The purpose of this study was to investigate the effect of ferric citrate nano-spray dried
particles on hydrogen production by using different concentrations of CG. From
previous investigation (section 2.4), 100 mg/L of ferric citrate particles was selected for
this investigation. Different concentrations of CG, viz., 100 mg/L, 500 mg/L, 1000 mg/L,
343
2000 mg/L, 5000 mg/L and, 10000 mg/L were separately distributed to different serum
bottles (125 mL). From a stock solution prepared using water, 100 mg/L of ferric citrate
particles was added to each bottle in such a way that after inoculating the bottle with 5%
(v/v) inoculum, total volume of the medium would become 25 mL. Similar to different
hydrogen production experiments already discussed, serum bottles were processed,
inoculated, incubated, and gas samples were collected, stored and analyzed (section
2.1 & 2.3).
Hydrogen analysis
Gas samples stored in 5.9 mL airtight glass vials were analyzed by gas
chromatography. The GC system used for this purpose was of Varian 3800 model
(USA), equipped with automatic sample injector. The detector was a thermal
conductivity detector (TCD) and the column used was PoraPLOT Q® (Agilent
technology, USA). For this analysis, N2 was used as the carrier gas and column
temperature was kept constant at 100 °C. The carrier gas flow rate of 3.5 mL/min was
used and the retention time for hydrogen was determined to be 4.5 min.
Results and Discussion
Characterization of nano-spray dried particles
SEM images of the ferric citrate particles used in the present investigation have been
presented in Figure 5.1.1. A very broad particle size distribution has been observed with
the diameters ranging from around 1.73 µm to 468 nm or smaller. Particles were
completely spherical and an aggregation tendency was observed among them. Zeta
potential distribution of these particles has been presented in Figure 5.1.2. From Figure
5.1.2, the zeta potential was determined to be more than ± 5 mV, which ruled out the
possibility of instant coagulation of the particles. However, it was lesser than ± 40 mV,
which implied that aggregation among the particles was possible. Therefore, the
observed aggregation behavior of ferric citrate particles can be considered as normal.
344
Screening of different nano-spray dried particles for biohydrogen production
NiFe-hydrogenase and FeFe-hydrogenase are two crucial enzymes responsible for
microbial hydrogen production in anaerobic environment (Kim et al., 2010, Marseille,
1998, Xu et al., 2013). The active site of NiFe-hydrogenase has one Ni and one Fe
atom; whereas, two Fe atoms are present in the active site of FeFe-hydrogenase
(Sarma et al 2014. Key Enzymes in Value-Addition of Glycerol to Biohydrogen. In:
Enzymes
in
value-addition
of
waste.
https://www.novapublishers.com/catalog/product_info.php?products_id=47561&osCsid=
cdc47dcd8e6201708571788e5dabc903
(accessed
on
22/02/2014).
Therefore,
hydrogen production media should contain sufficient amount of Ni and Fe, so that
synthesis of these enzymes are not affected. In CG, only a negligible amount of Ni may
be present; whereas, the amount of Fe presents in CG could be as low as 31.6 ± 19.0
ppm to 44.41 ± 16 ppm (Sarma et al., 2013). For hydrogen production by crude glycerol
bioconversion, the feedstock needs to be diluted to an optimum concentration; which is
usually around 10 g/L (Sarma et al., 2013). Hence, final concentration of Fe in a
biohydrogen production medium prepared using only CG (10 g/L) will be as low as 0.31
to 0.44 ppm. In this context, CG supplemented with Ni and Fe might have beneficial
effect on biohydrogen production by CG bioconversion. Therefore, the purpose of the
present study was to evaluate the effect of ferric citrate, FeSO4, nickel acetate, and
NiCl2 supplements supplied in nano-spray dried form, on hydrogen production by CG
bioconversion. In Figure 5.1.3 cumulative hydrogen production by bioconversion CG
supplemented with different nano-spray dried particles has been presented. As shown
in Figure 5.1.3, presence of relatively small amount of different particles in the media
was found to significantly alter the hydrogen production ability of the microorganism. As
there are different probable mechanisms for nickel toxicity in microorganisms; nickel
may be toxic to the bacterium used in this investigation (Macomber et al., 2011). It could
be a reason for reduced hydrogen production in the case of nickel acetate, and NiCl2.
Compared to nickel containing particles, more hydrogen production has been observed
in the case of iron containing particles. Between iron containing particles, particles
made up of ferric citrate were found to produce more amount (nearly 2.5 folds more) of
345
hydrogen (Figure 5.1.3) than FeSO4 particles. Citrate moiety of ferric citrate containing 6
carbon and 5 hydrogen atoms might have served as an additional/easily accessible
substrate for the process and it could be a reason for improved hydrogen production.
During fermentation some bacteria can use Fe (III) as electron acceptor (Pham et al.,
2003); thus ferric citrate might have additional beneficial role for the process. Based on
this observation, ferric citrate particles have been selected for further investigation.
Effect of Fe-citrate particle preparation method on hydrogen production
The purpose of the present investigation was to compare the effect of hot water based
and NaOH based nano-spray dried ferric citrate particles on hydrogen production.
Different amounts of these particles, ranging from 10 mg/L to 500 mg/L were tested as
supplement for hydrogen production by CG bioconversion and the results have been
presented in Figure 5.1.4. From Figure 5.1.4, it is clear that compared to ferric citrate
particles prepared using NaOH, hot water based particles are more favorable as a
supplement for hydrogen production. As shown in Figure 5.1.4, corresponding to
increased concentration of NaOH based ferric citrate from 10 mg/L to 500 mg/L,
decreased hydrogen production has been observed. In addition to ferric citrate, NaOH
based particles may also contain Na+ ions in large number. Therefore, unfavorable ionic
strength of the medium supplemented with such particle could be one reason of
observed reduced hydrogen production. On the contrary, by increasing the
concentration of hot water based ferric citrate particles from 10 mg/L to 500 mg/L,
hydrogen production was increased by nearly 50.45 % (Figure 5.1.4). Based on this
observation, 100 mg/L of hot water based ferric citrate particles have been selected for
further investigation. If relatively higher concentration of the supplement (ferric citrate
particles) is used, medium pH will decrease and presence of higher concentration of the
particles might have negative effect on the microorganisms used for hydrogen
production. Thus, choosing a relatively lower concentration of the particles will be an
appropriate option.
346
Effect of CG concentration on H2 production using media supplemented with
nano-spray dried ferric citrate particles
The purpose of this experiment was to determine the effect of initial CG concentration
on hot water based ferric citrate particle supplemented hydrogen production process.
Results of this investigation have been presented in Figure 5.1.5 and from the figure it
can be inferred that at constant ferric citrate particle concentration (100 mg/L), by
increasing the initial CG concentration from 100 mg/L to 10 g/L, cumulative hydrogen
production can be increased by nearly 12 folds. It is quite obvious as optimum CG
concentration for a batch hydrogen production process has been found to be around 10
g/L (Sarma et al., 2013).
As shown in Figure 5.1.3, however, if total hydrogen production by bioconversion of 1
kg of CG is considered; the amount projected to be produced at initial CG concentration
of 100 mg/L was found to be nearly 8.20 folds more than that of 10 g/L. In the case of
100 mg/L CG, the amount of ferric citrate particles (100 mg/L) added to the process was
equal to that of substrate (CG). Therefore, for hydrogen production media prepared
using one kg of CG, nearly 1 kg of ferric citrate particles would be needed. However, if
an initial CG concentration of 10 g/L is used and 100 mg/L of the particles is added as
supplement; for 1 kg of CG, only 10 g of the particles would be required. Therefore, 8.20
folds increase in hydrogen production projected for 100 mg/L CG supplemented with
100 mg/L of ferric citrate particles could be attributed to the additional amount of the
particles required for the approach.
In Figure 5.1.6, cumulative hydrogen productions by only CG (100 mg/L); CG (100
mg/L) supplemented with ferric citrate (100 mg/L) as well as CG (100 mg/L)
supplemented with nano-spray dried ferric citrate particles (100 mg/L) have been
presented. Figure 5.1.6 demonstrates that by supplementing the CG bioconversion
process with ferric citrate or its nano-spray dried particles, 17.18% to 31.71 % percent
improvement in hydrogen production can be achieved. However, supplementation
process will involve additional process cost; hence, a proper cost benefit analysis of the
approach is a prerequisite for its industrial trial. Moreover, it should also be determined,
347
whether the application of ferric citrate or its nano-spray dried particle will be
appropriate for large scale hydrogen production using CG. Likewise, process mode and
hydrogen partial pressure in the headspace of the reactor are known to have influence
on cumulative hydrogen production. Thus, in order to determine the potential of the
approach, unlike the closed batch process used in the present investigation, hydrogen
production using CG supplemented with ferric citrate or its nano-spray dried particle
should also be evaluated using process with proper pH control and at relatively low
hydrogen partial pressure.
A summary of different reports on hydrogen production by microbial conversion of crude
glycerol have been presented in Table 5.1.1. From the table, it is evident that compared
to reported maximum hydrogen yield by dark fermentation of CG (8.25 mol-H2/kg CG);
by using an initial CG concentration of 100 mg/L with no supplementary nutrient, 2.75
folds more hydrogen (22.7 mol-H2/kg CG) could be produced. This observation
indicated that hydrogen production by using an initial substrate concentration as low as
100 mg/L can significantly improve cumulative hydrogen production by maximum
feedstock utilization. This approach may enhance hydrogen yield not only in the case of
CG bioconversion but also for any other substrate. Three possible reasons for this
observed improved hydrogen yield are discussed below. (i) Volatile fatty acids (VFAs),
such as acetate, butyrate etc., are produced as by-products of fermentative hydrogen
production (Oh et al., 2003). Production of these byproducts can quickly decrease the
pH of the medium to trigger a metabolic shift, which may lead to decreased cumulative
hydrogen production (Khanal et al., 2004). At low initial substrate concentration (100
mg/L), lesser amount of these VFAs will be produced; hence, the concentration of these
compounds in the medium will be lower than a process with high initial substrate
concentration. Thus, inhibitory effect of VFAs could be avoided if a relatively low initial
substrate concentration is used. This, approach has industrial significance as it may
reduce the requirement of large amount of alkali and acid for process pH control. (ii)
Hydrogen partial pressure buildup in the headspace of the reactor can reduce
cumulative hydrogen production and by periodically flushing out the accumulated gas,
hydrogen production can be increased (Ngo et al., 2011). If an initial substrate
348
concentration as low as 100 mg/L is used, only a small amount of hydrogen will be
produced per liter of medium; therefore, hydrogen accumulation in the headspace will
be far lower than a process with high initial substrate concentration such as, 10 g/L.
Thus, low partial pressure of hydrogen in the headspace could be one of the reasons of
improved hydrogen yield per kg of substrate observed in the present investigation. (iii)
In the case of present investigation, initial CG concentrations ranging from 100 mg/L to
10 g/L were used; however, inoculum size was kept constant at 5 % (v/v). Therefore, in
the case of the process with 100 mg/L of CG, the amount of active microbial biomass
introduced per kg of substrate (CG) was 100 times more than that of the process with
10 g/L CG. Thus, the role of excess amount of biomass is undeniable.
Conclusions
Among ferric citrate, FeSO4, NiCl2 and Ni-acetate nano-spray dried particles, only ferric
citrate particles were found to have beneficial effect on hydrogen production. Two
different types of ferric citrate particles were prepared, either by dissolving it in hot water
or in NaOH solution. Between these two particles, hot water based ones were found to
enhance the hydrogen production by 50.45 %. Apart from supplementation, initial
substrate concentration has been identified as a crucial factor to determine the
hydrogen yield. At extremely low initial CG concentration of 100 mg/L, by supplementing
the process with ferric citrate particles, a hydrogen yield as high as 29.9 mol-H2/kg CG
has been achieved. This yield is significantly higher than the value of 8.25 mol-H2/ kg
CG; previously known for dark fermentative hydrogen production.
Acknowledgments
349
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352
Table 5.1. 1: Summary of different reports on hydrogen production by crude glycerol bioconversion
SL.
Fermentation
No.
type
1
Substrate
H2
H2 production
substrate
production
(mol/kg CG)
concentration
(reported)
Rhodopseudomo
RCV
70-75 % glycerol
0.92 g/L (10
nas
biotin,
mM) glycerol
CG
41
containing
%
(w/w)
Enterobacter
10 g/L glycerol
aerogenes
CG
with
86%
CG
containing
nearly
1.0%
6
mol/mol
Complex
HU-
medium,
phosphate buffer
0.71
mol-
H2/mol-
10 g/L glycerol
aerogenes ATCC
with
13048
agents
medium
Thermotoga
2 g/L yeast extract,
2.73
neapolitana DSM
0.05M
mol-H2 / mol-
4359
buffer
pretreated CG
(w/v) glycerol
(SabourinProvost
et
3.16 mol-H2/kg
(Ito et al.,
CG*
2005)
glycerol
Complex
g/L
mol-
Reference
al., 2009)
Enterobacter
5
48.86
H2/kg CG*
glycerol
CG containing
Dark
fermentation
para-
101
(w/w) glycerol
4
medium,
aminobenzoic acid
palustris
CG containing
glycerol
3
Supplement
CG containing
fermentation
2
Microorganism
containing
CG
Photo-
Initial
medium
624.8
mL-
8.25 mol-H2/kg
(Marques
buffering
H2/256
mL-
CG*
et al., 2009)
0.14
0.296
HEPES
±
mol-
(Ngo et al.,
H2/kg CG*
2011)
3.22 mol-H2/kg
(Sakai
CG*
al., 2007)
glycerol
consumed
5
CG
45
containing
%
(w/w)
CG containing
Enterobacter
NBRC
14.8g/L
aerogenes NBRC
medium, thionine
353
702
0.66
H2/mol-
mol-
et
SL.
Fermentation
No.
type
Substrate
Initial
glycerol
H2
H2 production
substrate
production
(mol/kg CG)
concentration
(reported)
glycerol
Microorganism
Supplement
12010
Reference
glycerol
consumed
6
CG
containing
3 g/L CG
Mixed culture
69.5 % glycerol
70 mM MES buffer,
0.31
nutrient solution
H2/mol-
mol-
2.33 mol-H2/kg
(Selembo
CG*
et al., 2009)
22.7 mol-H2/kg
Present
CG
study
29.9 mol-H2/kg
Present
CG
study
glycerol
7
CG
23.63
containing
±
100 mg/L CG
2.5%
CG
23.63
containing
±
2.5%
(w/w) glycerol
No supplement
aerogenes NRRL
(w/w) glycerol
8
Enterobacter
2.27
mmol-
H2/L
B-407
100 mg/L CG
Enterobacter
100
mg/L
aerogenes NRRL
citrate NP
ferric
2.99
mmol-
H2/L
B-407
*Calculated from literature data by assuming complete consumption of glycerol present in a CG based medium.
354
Figure 5.1. 1: SEM images (at different magnification) of ferric citrate particles prepared using hot
water
Zeta Potential Distribution
120000
Total Counts
100000
80000
60000
40000
20000
0
-100
0
100
Zeta Potential (mV)
Figure 5.1. 2: Zeta potential distribution of ferric citrate particles prepared using hot water
356
200
40
35
30
25
20
15
10
5
0
Fe-citrate
FeSO4
Ni-acetate
NiCl2
respective nanospray dried particles were added to 12 g/L of CG
357
45
40
35
30
25
NaOH
20
Hot water
15
10
5
0
10 mg/L
50 mg/L
100 mg/L 300 mg/L 500 mg/L
Figure 5.1. 4: Cumulative hydrogen production obtained after 48 h of incubation. Different
concentrations of two different types of ferric citrate particles were added to 12 g/L of CG
358
30
40
mol/kg
35
mmol/L
30
25
25
20
20
15
15
10
10
5
H2 production (mol/kg CG)
35
5
0
0
100 mg/L 500 mg/L
1g/L
2g/L
5g/L
10 g/L
Initial CG concentration
Figure 5.1. 5: Cumulative hydrogen production obtained after 48 h of incubation. 100 mg/L of ferric
citrated particles (prepared with hot water) were added to different concentrations of CG.
Projected hydrogen yields by bioconversion of 1 kg of CG have also been shown for each initial
CG concentration
359
3.5
3
2.5
2
1.5
1
0.5
0
Fe-citrate NP
Fe-citrate
Control
Figure 5.1. 6: Cumulative hydrogen production obtained after 48 h of incubation. Control is only
CG (100 mg/L)
360
CHAPTER 6
POTENTIAL APPLICATION OF ORGANIC ACID RICH LIQUID WASTE
FROM HYDROGEN PRODUCTION PROCESS FOR PHOSPHATE
SOLUBILIZATION
Chapter 6. Potential application of organic acid rich liquid waste
PART I
LIQUID WASTE FROM BIO-HYDROGEN PRODUCTION - A
COMMERCIALLY ATTRACTIVE ALTERNATIVE FOR PHOSPHATE
SOLUBILIZING BIO-FERTILIZER
a
Institut national de la recherche scientifique, Centre - Eau Terre Environnement, 490,
Rue de la Couronne, Québec(QC), Canada G1K 9A9
b
[email protected])
International Journal of Hydrogen Energy (2013) 38 (21): 8704–8707
363
Résumé
Au cours de la production de H2 par fermentation sombre des acides organiques
différents sont produits comme des sous-produits indésirables. Ces rejets liquides,
riches en acides organiques, ont une excellente capacité de solubilisation du phosphate
(augmentation de 36,1 fois) et pourraient être considérés comme une alternative
potentielle pour les bio-engrais commerciaux solubilisateurs de phosphate (PSB). Les
acides organiques produits par les bioengrais commerciaux solubilisateurs de
phosphate (PSB) sont reconnus pour chélater les cations de phosphates insolubles
(comme Ca3(PO4)2) présents dans le sol, favorisant la solubilisation du phosphate et le
rendre disponible pour les plantes.
Il est connu que le GB généré lors de la fabrication du biodiesel pourrait être utilisé
comme substrat pour la production d'hydrogène. Le déchet secondaire riche en acides
organiques pourrait être utilisé comme agent de solubilisation du phosphate. Ainsi, cette
nouvelle approche pourrait: (i) offrir un engrais vert; (ii) améliorer la viabilité économique
de la production de biohydrogène et; (iii) devenir un apport financier pour les fabricants
de biodiesel en leur offrant une stratégie de gestion des déchets rentable.
Mots-clés: biodiesel; biohydrogène déchets liquides; glycérol brut; acide organique;
phosphate solubilisation bioengrais; gestion des déchets
364
Abstract
Organic acids produced by commercial phosphate solubilizing bio-fertilizer (PSB) can
chelate the cations of insoluble phosphates (such as Ca3 (PO4)2) present in soil to
solubilize the phosphate and make it available for plants. Meanwhile, during H2
production (dark fermentation) different organic acids are produced as unwanted byproducts. Interestingly, the acid-rich liquid waste of the process has excellent phosphate
solubilizing ability (36.12 folds increase) and it could be considered as a potential
alternative for PSB. Studies have demonstrated that biodiesel manufacturing waste
(crude glycerol) could be used as a substrate for hydrogen production and the
secondary waste could be used as a phosphate solubilizing agent. Thus, this novel
approach could: (i) offer a green fertilizer; (ii) improve economic sustainability of biohydrogen production and; (iii) become a financial booster for biodiesel manufacturers by
offering them a profitable waste management strategy.
Keywords: biodiesel; bio-hydrogen liquid waste; crude glycerol; organic acid;
phosphate solubilizing bio-fertilizer; waste management
365
Introduction
Hydrogen is a renewable energy carrier which is postulated to be one of the clean fuels
of future (http://www.alternative-energy-news.info/technology/hydrogen-fuel/ (accessed
on 26/04/2013)). Water is the only by-product of its combustion and hence, it has the
potential to significantly reduce the greenhouse gas emission. As a fuel it is suitable for
fuel cells or internal combustion engines and energy content of H2 is higher than any
other gaseous fuels of equivalent weight (Meher Kotay et al., 2008). During biohydrogen production by dark fermentation, organic acids such as acetic, butyric and
lactic acid are produced (Shida et al., 2009), which are generally considered as waste.
Actually, production of organic acid can drop the pH of the medium to terminate the
process. Similarly, conversion of a significant portion of the substrate to different organic
acids results in reduction of H2 yield. Therefore, organic acid production is considered
as
the
major
bottleneck
of
dark
(http://www.slideshare.net/kumarhukkeri/finalppt-em-1
fermentative
(accessed
H2
on
production
02/03/2013)).
Interestingly, some of the organic acids can chelate the cations of insoluble mineral
phosphates (present in soil) and release soluble phosphate to make it available for
plants (Kucey, 1983). In fact, phosphate solubilization by production of organic acid is
the main plant growth promoting mechanism (Vyas et al., 2009) of commercially used
PSB. Thus, organic acid rich bio-hydrogen production wastes could be a potentially less
expensive alternative for PSB. Moreover, PSBs have relatively shorter shelf-life as they
are actually a preparation of living microorganisms and the organisms must be alive
when it is applied to the field. Alternatively, the performance of the proposed fertilizer
does not depend on the number viable cell it contains and hence, an extended shelf-life
could be expected. Thus, production, packaging, storage and application of proposed
fertilizer should be easier than conventional PSBs.
In general, the liquid waste of dark fermentative H2 production using any substrate/any
microorganism should be suitable as the proposed fertilizer. If the proposed strategy
could be integrated into an industrial process, such as biodiesel production, apart from
hydrogen production, it could become a profitable strategy for waste management. For
366
example, as presented in Figure 6.1.1, crude glycerol (CG), the major by-product (10 %
by weight) of biodiesel production process (Chatzifragkou et al., 2012) could be used
(as a substrate) for bio-hydrogen production and the final liquid waste could be used as
an alternative of phosphate solubilizing bio-fertilizers. In fact, crude glycerol is a
hazardous waste and its proper disposal is an additional expense for biodiesel
manufacturers. Therefore, if successful, the proposed approach may be a huge
breakthrough for financial/environmental sustainability of biodiesel industry.
Hydrogen production
Dark fermentative hydrogen production experiments were carried out using 10 g/L crude
glycerol (Rothsay, Canada) as the only media component. Actually, 10 g/L crude
glycerol was found to be optimum for hydrogen production (Sarma et al., 2012, Sarma
et al., 2013). Likewise, Enterobactor aerogenes NRRL B 407 (ARS, USDA, USA) was
used in this study and all other conditions of this investigation (H2 production) was
similar to our previous report (Sarma et al., 2012). Briefly, for each experiment, 50 mL
CG solution (10 g/L) was taken in 125 mL serum bottle and anaerobic condition was
created by passing ultrapure N2 gas through the medium. The bottles were properly
sealed (using aluminum crimp seal having silicon septum) and autoclaved. Finally, they
were inoculated using 5% (v/v) fresh Enterobactor aerogenes culture and incubated
(150 rpm/30°C). Unlike the previous study, in the present case, H2 gas produced during
the process was not periodically released form the serum bottles used for this purpose
(Sarma et al., 2012).
Phosphate solubilization study
To determine phosphate solubilization potential of the spent media of bio-hydrogen
production process, experiments were conducted using 6 different conical (glass) flasks
(125 mL). In each flask, 50 mg of Ca3 (PO4)2 (tricalcium phosphate) was taken and 50
mL of 0 %, 10 %, 20%, 40%, 60% and 100 % (v/v) of the spent media of crude glycerol
based bio-hydrogen production process were added (1 g Ca3 (PO4)2 in 1L liquid).
Finally, the flasks were incubated (150 rpm and 28° C) in an orbital incubator shaker
367
(Infors HT, Switzerland) and liquid samples were collected at 3 h interval for the first 12h
(and final samples were collected after 10 d).
Analysis
Gaseous samples collected during the investigation were analyzed by a gas
chromatograph (Sarma et al., 2012). Likewise, to determine the soluble phosphate
concentration, 1 mL of aqueous sample collected during the phosphorus solubilization
study was centrifuged (5000 g for 10 min.) and the suspended tricalcium phosphate
present in the solution was removed. About 0.1 mL of the supernatant was collected,
diluted 10 times with distilled water and soluble phosphate present in the solution was
analyzed by using a method described by (Murphy et al., 1958). As described by the
authors, all glass-wares used in the analysis were pretreated with concentrated H2SO4
(Murphy et al., 1958).
Cumulative H2 production using 10 g/L crude glycerol as the only substrate was found
to be 20.61 mmol/L (H2 production profile is not shown). This value is lower than our
previous finding (84.08 mmol/L) using same concentration of the crude glycerol. It is
well known that process mode can strongly influence the hydrogen yield and the
observed difference is due to two variable process modes adopted. In the present
case, a closed batch system was used where H2 produced during the process was
accumulated in the headspace of the vessel, whereas, in previous case, produced gas
was periodically released from the headspace. Increase in H2 partial pressure in the
headspace is known to negatively affect the production and hence the final
concentrartion in the two cases were different.
Liquid waste generated during crude glycerol bioconversion and H2 production process
has been evaluated for phosphate solubilization potential. Figure 6.1.2 summarizes the
results of phosphate solubilization study involving the bio-hydrogen production waste. In
fact, soluble phosphate concentration of the spent medium itself was found to be 14.96
mg/L. As a control experiment, when only distilled water (0% (v/v) of the waste) was
used, soluble phosphate concentration was found to be only 1.38 mg/L. In contrast,
368
soluble phosphate concentration as high as 32.06 mg/L had been achieved by using
bio-hydrogen production waste (100 %, v/v), which is 23.17 folds higher than the control
experiment. This finding (23.17 folds increase within 1 minute) is significant in a
situation where conventional PSBs were known to liberate similar amount of phosphate
in a period of 2-3 days (Trivedi et al., 2008). As shown in Table 6.1.1, (Zhu et al., 2011)
have reported that a newly isolated strain of phosphate solubilizing bacteria can
solubilize 283.16 mg/L of phosphorus within an incubation period of 11 days. However,
it should be noted that the authors performed the phosphate solubilization study using
chemically defined liquid media in optimum laboratory conditions. Hence, the
performance of the strain could be different in real field application. Moreover, during
storage, the cell count of a biofertilizer formulation can sharply decrease (Menaka et al.,
2007) and further reduce its phosphate solubilization ability. Thus, considering the fact
that in the case of biohydrogen production, liquid waste phosphate solubilization is
instant as well as it has nothing to do with microbial cell viability, performance of the
present approach is comparable to that of a conventional PSB. A detailed comparison
of the two fertilizers has been presented in Table 6.1.1. Likewise, the phosphate
solubilization processes was monitored at 3 h interval for 12h, and phosphate
solubilization achieved within 1 minute was only slightly different to that observed after
12h (data not shown). However, after 10 d of incubation enhancement in cumulative
soluble phosphate concentration was recorded to be as high as 18.59, 25.86 and 36.12
folds for 40, 60 and 100 % (v/v) of the waste, respectively. Interestingly, any significant
improvement in soluble phosphate concentration was not observed for 10 and 20 %
(v/v) of the waste. One possible explanation for such observation could be that a small
number of hydrogen producing bacteria (facultative anaerobe) were still active in the
spent media and they played certain role (e.g. production of organic acid or enzyme
such as phosphatase) in phosphate solubilization. In the case of 10 and 20 % (v/v) of
the waste, such microbial activities were not sufficient (assuming that only a small
fraction of the bacteria was active) for a significant response. Moreover, for these two
cases, a slight decrease in soluble phosphate concentration was observed. It might be
due to incorporation of bioavailable phosphate into the biomass (synthesis of DNA,
369
RNA, phospholipids etc.). However, these are simple assumptions and further
investigation is required to verify them. Thus, it could be concluded that bio-hydrogen
production waste has excellent potential as a phosphate solubilizing agent.
In general, a commercial PSB is capable of increasing the phosphate availability by 2530 kg per hectare and it is sufficient to increase the product yield up to 20% (Bionics
Technology,
New
Delhi,
India.
http://www.indiamart.com/bionics-
technology/fertilizers.html (accessed on 21/02/2013)). Meanwhile, according to the
estimated hydrogen production, liquid waste generated by bioconversion of 1 kg of
crude glycerol would be sufficient to liberate 20 g of phosphate. Hence, for each
hectare, waste generated from 1250 kg of CG will be required to completely replace a
commercial PSB (Table 6.1.1). Therefore, for convenience of application, preconcentration of the waste may be required. Although, phosphate solubilizing ability of
bio-hydrogen production waste has been demonstrated its actual plant growth
promoting potential needs to be verified by field scale experiment using food crop.
Conclusions
Bio-hydrogen production liquid waste is an excellent phosphate solubilizing agent and it
has high potential to be a commercially important alternative of phosphate solubilizing
bio-fertilizer. It can increase the soluble phosphate concentration by 23.17 folds within 1
minute and 36.12 folds after 10 d (of incubation) of its application to a liquid system and
this value may be even better depending on the hydrogen production process adopted.
Unlike traditional phosphate solubilizing bio-fertilizers, the proposed alternative is not a
formulation of living microorganisms and hence, it is postulated to have superior shelflife. Finally and most importantly, the proposed fertilizer production process could be
integrated to biodiesel manufacturing process (aka biorefinery) to upgrade the biodiesel
industry to a “high profit-zero waste” industry.
370
Acknowledgments
SJ Sarma is thankful to FQRNT for providing financial assistance through a doctoral
fellowship (MELS). The authors are also thankful to NSERC, CRIQ and INRS-ETE
Canada for financial support.
371
References
Chatzifragkou A & Papanikolaou S (2012) Effect of impurities in biodiesel-derived waste
glycerol on the performance and feasibility of biotechnological processes.
Applied microbiology and biotechnology 95(1):13-27.
Kucey R (1983) Phosphate-solubilizing bacteria and fungi in various cultivated and
virgin Alberta soils. Canadian Journal of Soil Science 63(4):671-678.
Meher Kotay S & Das D (2008) Biohydrogen as a renewable energy resource—
prospects and potentials. International Journal of Hydrogen Energy 33(1):258263.
Menaka V & Alagawadi A (2007) Impact of inoculum levels on survival of phosphate
solubilizing bacterium on seeds and its performance in groundnut. Karnataka
Journal of Agricultural Sciences 20(1):65.
Murphy J & Riley J (1958) A single solution method for the determination of soluble
phosphate in sea water. J. Mar. Biol. Assoc. UK 37:9-14.
Sarma SJ, Brar SK, Le Bihan Y, Buelna G, Rabeb L, Soccol CR, Naceur M & Rachid B
(2012) Evaluation of different supplementary nutrients for enhanced biohydrogen
glycerol. International Journal of Hydrogen Energy 38 (5): 2191-2198.
and Biotechnology 88 (12): 2264–2271.
Sethi SK & Adhikary SP (2012) Cost effective pilot scale production of biofertilizer using
Rhizobium and Azotobacter. African Journal of Biotechnology 11(70):1349013493.
Shida GM, Barros AR, Reis CMd, Amorim ELCd, Rissato Zamariolli Damianovic MH &
Silva EL (2009) Long-term stability of hydrogen and organic acids production in
372
an anaerobic fluidized-bed reactor using heat treated anaerobic sludge inoculum.
Trivedi P & Sa T (2008) Pseudomonas corrugata (NRRL B-30409) mutants increased
phosphate solubilization, organic acid production, and plant growth at lower
temperatures. Current microbiology 56(2):140-144.
Vyas P & Gulati A (2009) Organic acid production in vitro and plant growth promotion in
maize under controlled environment by phosphate-solubilizing fluorescent
Pseudomonas. BMC microbiology 9(1):174.
Zhu F, Qu L, Hong X & Sun X (2011) Isolation and characterization of a phosphatesolubilizing halophilic bacterium Kushneria sp. YCWA18 from Daqiao Saltern on
the coast of yellow sea of China. Evidence-Based Complementary and
Alternative Medicine 2011.
373
Table 6.1. 1: Possible benefit of proposed technology compared to conventional PSB
Fertilizer
Phosphate
Phosphate
Amount
Chemicals
Shelf-life
Production
Additional
Pollution/GHG
solubilization
solubilization
required to
required
Cost
benefit
emission
ability
time
solubilize 25
(other than
kg phosphate
being a
(for one
sustainable
hectare of
fertilizer)
land)
Conventional
283.16 mg/L
Long
Equivalent to
Yes,
Only 6
Includes
PSB
media (after
incubation
88339.22 L of
expensive
months as it
fermentation
waste may need
11days of
period (10 to
fermented
chemicals
is a
cost (around
separate
incubation)*
30 days)
broth
as media
formulation of
$0.25/L)***
treatment
components
living
and
microorganis
formulation
m**
cost
No
Manufacturing
Bio-
49.98 mg/L
Instant
Bio-hydrogen
No, only
Expected to
No
Production of
Zero waste; high
hydrogen
waste
solubilization
production
H2O and CG
be superior
production
carbon free
GHG emission
production
waste
required
as it is not a
cost (waste
biofuel
reduction
waste
generated from
formulation of
by-product)
1250 kg of CG
living
additional
microorganis
biofuel
m
production
* (Zhu et al., 2011), **as for average bio-fertilizer, *** (Sethi et al., 2012)
374
potential due to
Figure 6.1. 1: A schematic representation of the proposed concept
375
Soluble phosphorus concentration (mg/L)
60
Within 1 minute of application
After 10 d of incubation
50
40
30
20
10
0
Control (0%)
10% (v/v)
20% (v/v)
40% (v/v)
60% (v/v)
100% (v/v)
Spent medium of bio-hydrogen production
Figure 6.1. 2: Phosphate solubilization achieved using different concentrations of bio-hydrogen
production liquid waste. The control represents soluble phosphate concentration obtained using
only water as the solubilizing agent
376
PART II
NOVEL ENVIRONMENTAL AND AGRICULTURAL USES OF
BIOHYDROGEN PRODUCTION WASTEWATER-A STUDY USING
SOYBEAN PLANTS
a
b
[email protected])
Journal of environmental management (Submitted)
377
Résumé
Avec l’absence d’émission de CO2 et une densité d'énergie massique plus élevée que
les carburants conventionnels, tels que l'essence, le diesel, le biodiesel et le bioéthanol,
le
biohydrogène est une option prometteuse en terme d'énergie renouvelable.
Cependant, la production commerciale de biohydrogène est principalement limitée par
le coût élevé des processus impliqués. En outre, au cours de la production d'hydrogène
par fermentation sombre, 60-70% de la charge d'alimentation est convertie en différents
sous-produits, dominés par des acides organiques. Afin de faire face à l'utilisation
inefficace des substrats ainsi que le coût élevé des étapes du procédé, une approche
simple de valorisation des déchets liquides de la production d'hydrogène (HPLW) a été
démontrée. Les microorganismes présents dans le sol peuvent produire des acides
organiques pour solubiliser les différentes formes de phosphates inorganiques, tels que
Ca3(PO4)2, en chélatant les cations et libérant ainsi le phosphore soluble. Ce phosphore
soluble peut être ensuite utilisé par les végétaux. De façon expérimentale, le rejet
HPLW riche en acide organique a été ajouté à un sol contenant des semences de soya.
Une augmentation de l'assimilation du phosphore par les plantes de soja de 2,2 à 2,7
fois a été observée.
Mots-clés: Biohydrogène; solubilisation du phosphore; acides organiques; rejets de
fermentation, soya
378
Abstract
With CO2 free emission and a gravimetric energy density higher than conventional fuels,
such as gasoline, diesel, biodiesel and bioethanol; biohydrogen is a promising option as
a green renewable energy carrier. However, commercial production of biohydrogen is
mostly limited by high process cost involved. Moreover, during hydrogen production by
dark fermentation, 60-70 % of the feedstock is converted to different byproducts,
dominated by organic acids. In order to cope with inefficient substrate utilization as well
as high process cost; a simple approach of value-addition of hydrogen production liquid
waste (HPLW) has been demonstrated. Phosphate solubilizing microorganisms present
in soil can produce different organic acids to solubilize inorganic phosphates, such as
Ca3 (PO4)2, by chelating the cations, and release soluble phosphorus to be utilized by
plants. Thus, organic acid rich HPLW has been added to soil and 2.18 to 2.74 folds
increase in phosphorus uptake by soybean plants has been observed.
Keywords: Biohydrogen; phosphorus solublization; organic acids; spent medium;
soybean plants
379
Introduction
Biohydrogen can be used as a CO2 free fuel, since its combustion releases only water
as major emission to the atmosphere. This property makes it more environmentally
friendly than commercial biofuels, such as biodiesel, bioethanol and biomethane.
Moreover, the gravimetric energy density of hydrogen is 142 MJ/kg; which is nearly 3
times of major fossil fuels, such as gasoline (47 MJ/kg), and diesel (43 MJ/kg) (Şensöz
et al., 2000, Sydney et al., 2014). Biohydrogen can be produced from a diverse group of
renewable raw materials including non-food crop based biomass and agro-industrial
organic wastes. Dark fermentation of these materials is relatively rapid, simple and most
commonly used technique for biohydrogen production. However, during this process,
only 30-40 % of the feedstock is actually used; rest is converted to different byproducts
(Venkateswar Reddy et al., 2014). These byproducts include mainly different volatile
fatty acides (VFAs), in the form of acetate (15.3-34.1%), butyrate (31.2-45.6%),
propionate (0.9-15.9%), lactate (2-4.6%) and alcohols such as ethanol (4.6-10.1%),
among others (Fang et al., 2002). Value addition of the hydrogen production liquid
waste (HPLW) containing these byproducts may give an additional revenue source to
commericial biohydrogen production; which is otherwise impared by high process cost.
In this context, as a part of present investigation, acid rich HPLW has been evaluated as
phosphate solubilizer for soybean cultivation.
By weight, phosphorus content of a typical soil is around 0.05%, of which, only around
0.1% is directly available to plants (Mardad et al., 2013). In soil, a significant amount of
phosphorus is present in its insoluble forms, such as Ca3 (PO4)2, CaHPO4, FePO4 and
AlPO4. Moreover, soluble phosphorus supplied to agricultural fields in the form of
chemical fertilizer, may react with cations, such as Ca2+, Fe3+, Al3+ to precipitate
immediately after its use and become unavailable (Mardad et al., 2013, Zhu et al.,
2012). Application of phosphate solubilizing biofertilizer (PSB) is a solution to this
problem. PSBs are microorganisms which can secret a range of organic acids in order
to chelate the cations of insoluble inorganic phosphate present in soil such as,
Ca3(PO4)2 and solubilize them to be available for plants. Thus, soil application of organic
380
acid rich HPLW has been postulated to have similar beneficial effect on phosphorus
uptake by plants.
For the present investigation, crude glycerol (CG) has been used as the feedstock for
hydrogen production by Enterobacter aerogenes. CG is a waste generated during
biodiesel manufacturing by transesterification of lipid (Pott et al., 2014, Zhou et al.,
2014). As high as 10 kg of CG may be generated for every 100 kg of biodiesel produced
(Santibáñez et al., 2011). Commercially CG is mainly used as a source of glycerol.
However, due to increase in global CG production, market price of glycerol has been
dropped by nearly 20 folds (Pott et al., 2013). Therefore, researchers are looking for an
alternative option for CG valorization. In this context, as shown in Figure 6.2.1, CG has
been used as a feedstock for hydrogen production. Liquid waste generated during this
process has been applied to soil as phosphate solubilizer and its effect on phosphorus
uptake by soybean plants has been determined.
Enterobacter aerogenes NRRL B-407 collected from ARS (USDA, USA) was used for
hydrogen production by CG bioconversion. It is a facultative anaerobic, gram-negative
rod-shaped bacterium, which belongs to Enterobacteriaceae family. For maintenance of
the strain, it was periodically grown in a complex medium composed of glucose (5 g/L),
casein peptone (5 g/L), KH2PO4 (2 g/L), yeast extract (0.5 g/L) and MgSO4.7H2O (0.5
g/L). About 50 mL of the medium was taken in serum bottle (125 mL), sparged with N2
gas for 2 minutes and closed with crimp seal having silicon septum. After sterilization,
these serum bottles were used to grow the microorganism. The incubation condition
routinely followed for growing the organism was 30°C and 150 rpm and an incubator
shaker was used for incubating the bottles.
For inocula preparation for the present investigation, serum bottles containing a medium
made up of 12 g/L of CG was used. Other conditions of media preparation and
incubation were similar to those mentioned above. After 24 h of incubation, 5 % (v/v) of
381
microbial culture were used as the inocula for hydrogen production experiments carried
out in a 7.5 L bioreactor.
Biohydrogen production from crude glycerol by using a 7.5 L bioreactor
Hydrogen production experiments were carried out in a 7.5 L bioreactor (Labfors 5,
Infors-HT, Canada). About 3 liters of medium containing 12 g/L of CG as the only
medium component was used for the present study. The bioreactor vessel along with
the medium was autoclaved at 121 ºC for 20 minutes. After cooling, ultrapure nitrogen
gas was passed through the medium to bring the dissolved oxygen concentration to
zero. pH of the medium was adjusted to 6.6 and inoculated with 5 % (v/v) inoculum,
which has been prepared as described in previous section (section 2.1). The
temperature of the bioreactor was maintained at 30 ºC and the agitation used for the
process was 200 rpm. The bioreactor was connected to a computer interfaced with
process control software (Iris, Infors-HT, Canada); the signals of different sensors were
recorded online. In order to record real time pH change of the process, after inoculation,
the automatic pH control was inactivated.
Investigation of phosphorus uptake by soybean plant in presence of HPLW
Soybean (Glycine max) was selected as the model plant for phosphorus uptake
behavior study and the seeds were collected from local market. Plastic pots containing
120 g of soil (Floral premium potting mix; Canadian tire) were used for the present
investigation. In Table 6.2.1, the amount of HPLW and tricalcium phosphate (Ca3(PO4)2)
mixed with the soil of different pots has been presented. A pot containing only soil (pot
3) was used as control for the experiment. 15 soybean seeds were placed in each pot
and subsequently covered with nearly 1 cm thick layer of soil. Distilled water was daily
provided to each pot to keep the soil sufficiently wet. All pots containing soybean seeds
were incubated in a plant growth chamber (Conviron®, Canada) maintained at 25 ºC.
Inside the chamber, the pots were exposed to constant illumination by using two 160
watt Philips tubes. After one month, plants were harvested and analyzed for phosphorus
content as explained in section 2.4.3.
382
Analytical techniques
Hydrogen analysis
In order to determine the hydrogen concentration in the biogas accumulated in the
headspace of the reactor, gas samples were collected by using a gastight syringe (SGE
Analytical Science, Australia), stored in 5.9 mL gas sampling vial (Exetainer®, Labco,
UK) and subsequently analyzed by gas chromatography. A Varian 3800 gas
chromatograph (Varian, USA), fitted with PoraPLOT Q® column (Agilent technology,
USA) and thermal conductivity detector (TCD) was used for this purpose. Ultra-pure
nitrogen at a flow rate of 3.5 mL/min was used as the carrier gas and the column
temperature was 100 °C (Sarma et al., 2014).
Characterization of HPLW
In order to identify the major byproducts of hydrogen production, at the end the process
liquid sample was collected from the fermenter and analyzed by gas chromatography.
Prior to the analysis, the sample was centrifuged for 5 minutes at 10000 rpm and the
supernatant was filtered by 0.45 µ syringe filter. The GC (GC7890B, Agilent
Technologies, USA) used for this analysis was fitted with HP-INNOWax column (Agilent
Technologies, USA) and a flame ionization detector (FID). The column temperature
gradient used for this analysis was 50-250ºC and the injection volume was 0.8 µl.
Isobutanol was used as the internal standard.
Phosphorus analysis
For this analysis, a standard method for persulfate digestion for total phosphorus
analysis (https://uwlab.soils.wisc.edu/files/procedures/DNR_TotalP.pdf (accessed on
02/04/2014)) was followed by a colorimetric method for phosphate determination
(Murphy et al., 1958, Sarma et al., 2013a). Briefly, a digestion solution was prepared by
dissolving 12.8 g ammonium persulfate in 32 mL of 5.6 M H2SO4 and total volume of the
solution was adjusted to 100 mL by adding Mili-Q water. Stem of the soybean plants
grown in different pots were cut into small pieces and 50 mg form the plant of each pot
were used to analyze the phosphorus content. The stem pieces were taken in a 15 mL
383
screw cap thick walled teflon acid digestion tube and 8 mL of water was added. 0.5 mL
of the digestion solution was added to each tube and tightly closed. The digestion tubes
containing the samples were autoclaved at 121ºC for 30 minutes. After cooling,
phosphorus concentration in the solution was determined as reported earlier (Sarma et
al., 2013a).
Measurement of biomass production
In order to investigate the effect of HPLW addition to soil on soybean plant biomass
production, plants were picked from all different pots. The roots were carefully washed
with soap solution as well as distilled water to remove the soil and spread on tissue
paper for two hours to dry. Finally, the plants were weighed by using a digital balance.
Determination of soil pH
After harvesting the plants, one g of soil was collected from each pot and dissolved in
20 mL of distilled water. The samples were thoroughly mixed by using a vortex mixture
and pH of the solutions was recorded by a pH meter.
Results
Biohydrogen production from crude glycerol
A 7.5 L bioreactor was used for hydrogen production by CG bioconversion. During
fermentation, changes in the values of different process parameters, such as pH,
dissolved oxygen concentration (DO), temperature, and agitation were recorded online.
These profiles have been presented in Figure 6.2.2a. From Figure 6.2.2a, it is evident
that other than pH, all parameters were kept constant throughout the fermentation
period. There was a sharp decrease in medium pH until nearly 48-50 h; and even after
that it continued to decrease at a slower rate until termination of the process. As already
mentioned, during hydrogen production by dark fermentation, different volatile fatty
acids (VFAs) are produced as byproducts (Sydney et al., 2014). Therefore, quick
decrease in medium pH indicates the accumulation of these products. A gas
chromatogram of HPLW has been presented in Figure 6.2.2b. The chromatographic
analysis showed that the HPLW contains acetic acid (734.59 mg/L), butyric acid (90.23
384
mg/L) as well as residual glycerol (retention time 11.403 min.). VFAs containing liquid
waste can solubilize inorganic phosphate such as tricalcium phosphate (Ca3(PO4)2) to
release soluble phosphate (Sarma et al., 2013a). Hence, the HPLW of present
investigation was tested as a phosphate solubilizer to facilitate phosphorus uptake by
soybean plants and the results were discussed in section 4.2.
Investigation of phosphorus uptake by soybean plant in presence of HPLW
Soybean plants grown in different pots containing soil or soil amended with HPLW or
tricalcium phosphate (TCP) are analyzed for phosphorus uptake during one month
growth period. Based on this analysis, phosphorus content per g of biomass was
calculated for different plant samples; which has been presented in Figure 6.2.3. As
shown in Figure 6.2.3, compared to control (only soil), the plants grown in soil amended
with 50 and 100 mL of HPLW, respectively, have shown 2.18 and 2.74 folds increase in
phosphorus uptake. From this observation, it is evident that HPLW is capable of
improving the phosphorus uptake behavior of soybean plants. It has been demonstrated
that HPLW can solubilize insoluble inorganic phosphate to release the orthophosphate
group; which is available for plants (Sarma et al., 2013a). Thus, improved phosphorus
uptake can be considered as a consequence of its increased bioavailability due to
HPLW’s presence in the soil.
Discussion
Biohydrogen production from crude glycerol
As mentioned in section 2.4.1, gaseous samples were collected from the headspace of
the reactor and analyzed by GC. At the end of the fermentation (120h), hydrogen
concentration in the headspace was found to be nearly 0.5 mmol/L-medium. Based on
this observation, certain approaches can be considered for further improvement in
hydrogen production. Firstly, glycerol is the major component of CG and hence, it is a
good carbon/energy source for fermentative hydrogen production. However, it has
relatively lower amount of other nutrients such as, nitrogen rich compounds, Mg, Fe,
among others (Sarma et al., 2013b). Therefore, introduction of additional nutrient to the
process; either by direct addition of supplements to the medium or by growing the
385
inoculum in a nutrient rich medium, further improvement in hydrogen production might
be possible. Similarly, if no buffering agents are used, hydrogen production medium
containing only CG has very low buffering capacity. Therefore, in the present case, a
sharp decrease in medium pH was observed. Metabolic shift is a well-known
phenomenon in fermentative hydrogen production which can reduce the product yield.
Accumulation of VFAs and change in media pH is one major factor which can initiate a
metabolic shift. Hence, change in media pH and subsequent metabolic shift could be a
reason of reduced hydrogen production observed in the present study. For improved
hydrogen production, therefore, further optimization of the process parameters is
needed.
Phosphorus uptake by soybean plant in presence of HPLW
As shown in Figure 6.2.3, for pot 5 the plants were found to uptake nearly 2.84 folds
more phosphorus in presence of 1 g of TCP; even in the absence of HPLW. These
observations indicate that although TCP is sparingly soluble in water (6.4 x10-25 µg/L); if
applied on soil at relatively high TCP: soil ratio of 1:120, a considerable amount may
become available to the plants. Additionally, slightly acidic pH of the soil might have
helped in solubilizing a portion of the inorganic phosphate; since literature data
suggests that application of insoluble rock phosphate may be helpful in improving the
soluble phosphorus level of acidic soil, although, it is unlikely for neutral and alkaline soil
(Kumar et al., 2001). Further, as shown in Figure 6.2.3, phosphorus uptake observed for
pot 1 and 5 was almost similar. This observation has ascertained that application of
phosphorus rich material to the soil can be avoided by the addition of HPLW and the
latter has the potential to increase the bioavailability of phosphorus already present in
soil.
Further, compared to the plants grown in soil containing TCP (pot 5); plants grown in
soil amended with both HPLW (50 mL) and TCP (pot 4) have shown 8.86 %
improvement in phosphorus uptake. However, it should be noted that in this case of pot
4 as high as 1 g of insoluble TCP was added to the soil and 50 mL of HPLW was used
as phosphate solubilizer; hence, expected phosphorus uptake was more than observed
386
uptake. This is more so because, in the case of pot 2, addition of only HPLW (50 mL)
was found to improve the phosphorus uptake by 2.18 folds (Figure 6.2.3). At the same
time, as shown in Figure 6.2.3, among all the plant samples analyzed, the plants of pot
4 have taken up maximum amount of phosphorus (43.34 µg/g biomass). This
observation indicates that in the case of pot 4, probably a considerable amount of
soluble phosphorus remained unutilized and unlike the present case, a growth period of
more than one month would be more appropriate to observe a significant difference in
phosphorus uptake between pot 4 and pot 5.
For each pot, biomass production was estimated after one month of growth period and
the results have been presented in Figure 6.2.4. Among all the pots containing soil with
different additives, best biomass production was observed in the case of un-amended
soil. In this context it should be considered that the soil used in the present investigation
was actually a commercial potting mix designed for gardening. Therefore, in spite of
observed better phosphorus uptake by the plants of other pots, the one containing only
soil was capable of supporting relatively better biomass production. If pot 1, 2 and 3 are
considered; from Figure 6.2.4 it is evident that addition of 100 mL of HPLW to the soil
resulted in reduced biomass production. Corresponding to a decrease of the amount of
HPLW from 100 to 50 mL; an increase in biomass production was observed. The
observation indicated that the HPLW has inhibitory effect on biomass production and
the inhibition can be reduced by decreasing the amount added. Therefore, the dose of
HPLW beneficial for the plants should be determined by further optimization. In this
context, it should be considered that HPLW was mixed with the soil at the beginning of
the experiment; hence, delicate seedlings were exposed to large amount of HPLW at an
early stage of growth. Therefore, there is a possibility that the plants will behave
differently if the HPLW is applied to the field once the plants are mature enough to grow
in its presence.
Interestingly, as shown in Figure 6.2.4, addition of 50 mL of HPLW was found to have
beneficial effect on seed germination (33.33% improvement). Similarly, 1 fold increase
in seed germination was observed for soil containing both TCP and 50 mL of HPLW.
However, negative effect on seed germination has been observed for 100 mL of HPLW.
387
It has been observed that presence of ethanol in the soil can improved the germination
rate in some cases (Salehi et al., 2008). Ethanol may present in HPLW as a byproduct
of light independent fermentative hydrogen production process (Rosa et al., 2014).
Additionally, HPLW also contains acetic acid (734.59 mg/L), butyric acid (90.23 mg/L)
and residual glycerol. Combine effect of these chemicals, therefore, could be a reason
of improved germination rate observed in the present study. Most probably, soybean
seeds were protected by these chemicals from harmful microbial attack, thereby,
increasing their chance to germinate. However, this hypothesis needs experimental
evidence.
Equal amount of HPLW (50 mL) was added to pot 2 and pot 4; however, relatively
higher biomass production has been observed in the latter case (Figure 6.2.4). As
already discussed, in addition to HPLW, 1 g of TCP was also mixed with the soil of pot
4. Thus, present observation indicates that in presence of sufficient amount of insoluble
inorganic phosphate, inhibitory effect of HPLW on biomass production is reduced. Most
probable reason for such observation could be the reaction between TCP and the
organic acids of HPLW, which might have neutralized the acids immediately after their
introduction to the soil to release soluble orthophosphate. Therefore, in order to avoid
the inhibitory effect of unused acids and for improved plant biomass production, the
dose of HPLW needs further optimization.
Over their growth period, phosphate solubilizing microorganisms present in rhizosphere
can produce small quantities of different organic acids, and in typical soil solution the
concentration of these acids may vary from 1 to 50 mM
(Mardad et al., 2013). It
indicates that if organic acids are applied to the agricultural field at relatively low
concentration, in terms of phosphate solubilization, they may be beneficial for the plants
and will have no negative consequences. HPLW may contain as high as 44.06 mM of
acetate, 31.03 mM of butyrate, 8.21 mM propionate and other VFAs in small quantities
(Foglia et al., 2011). Therefore, instead of applying large amount of HPLW at the
beginning of cultivation, its multiple small doses over the growth period of plant could be
a viable option for reducing its potential inhibitory effect and for maintaining its
phosphate solubilization efficiency. Alternatively, similar to controlled drug delivery
388
(Brannon-Peppas, 1995), a carrier can be designed for sustained delivery of these
organic acids to agricultural soil. However, such approach needs proper cost benefit
analysis before considering.
After harvesting the plants, the pH of the soil of different pots was measured. It has
been observed that soil pH was almost constant even after addition of the organic acid
rich HPLW (data not shown). Therefore, the possibility of decreasing the soil pH post
HPLW application was negligible. Thus, HPLW could be a good alternative of
phosphate solubilizing biofertilizer. In fact, it has been postulated to be lesser expensive
as well as have better shelf life than commercial phosphate solubilizing biofertilizers
(Sarma et al., 2013a). The present investigation revealed that HPLW can improve
phosphorus uptake by plants; however, its positive effect on the yields of agricultural
products needs to be demonstrated by detailed investigation.
In conclusion, nearly 0.5 mmol-H2/L-medium has been produced by CG bioconversion
in the absence of any supplementary media components. Further optimizations of
different process parameters are needed to improve the hydrogen yield. Acetic acid
(734.59 mg/L) and butyric acid (90.23 mg/L) were the major byproducts of this process.
The liquid waste containing these acids has been evaluated as soil phosphate
solubilizer. Soybean plants grown in this liquid waste enriched soil have shown 2.18 to
2.74
folds
more
phosphorus
uptake.
The
observation
suggested
possible
commercialization of hydrogen production liquid waste as an alternative of phosphate
solubilizing biofertilizer. However, further field scale investigations are required to
optimize the approach.
Acknowledgments
389
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392
Table 6.2. 1: Soil composition of different pots used to grow soybean plants (HPLW, hydrogen
production liquid waste; TCP, Tricalcium phosphate).
Pot number
Soil (g)
HPLW (mL)
TCP (g)
Pot 1
100
0
Pot 2
50
0
0
0
Pot 4
50
1
Pot 5
0
1
Pot 3 (control)
120
393
Figure 6.2. 1: A schematic representation of the approach considered for the present
investigation. E-1 (Lipid), E-2 (Methanol), E-3 (NaOH/catalyst), E-4 (Transesterification), E-5
(Biodiesel processing), E-6 (Pump), E-7 (Biodiesel distribution), E-8 (Crude glycerol fermentation),
E-9 (Organic acid rich liquid waste), E-10 (Agricultural field), E-11 (Gas separator), E-12 (Gas
holder-CO2), E-13 (Gas holder-H2), E-14 (Compressor), E-15 (H2-cylinders); P (Pipeline); V (Valve).
394
Figure 6.2. 2: (a) Values of different process parameters recorded online during hydrogen
production using a 7.5L fermenter. (b) Gas chromatogram of hydrogen production liquid waste
(HPLW) showing the presence of acetic acid and butyric acid as major byproducts.
395
Phosphorus (µg/g biomass)
50
45
40
35
30
25
20
15
10
Figure 6.2. 3: Phosphorus uptake by soybean plants cultivated in the presence of different
amounts of hydrogen production liquid waste (HPLW) and tricalcium phosphate (TCP).
396
12
10
40
Biomass
35
Germination %
30
8
25
6
20
4
15
2
10
0
5
Germination %
Total biomass/10 plants (g)
Figure 6.2. 4: Effect of hydrogen production liquid waste (HPLW) and tricalcium phosphate (TCP)
treatment of soil on soybean plant biomass production and seed germination.
397
CHAPTER 7
DESIGNING AND OPTIMIZATION OF NOVEL BIOPROCESSES FOR
IMPROVE HYDROGEN PRODUCTION USING CG
Chapter 7. Designing and optimization of novel bioprocesses
PART I
A NOVEL ANAEROBIC TWO PHASE SYSTEM FOR BIOHYDROGEN
PRODUCTION AND IN SITU EXTRACTION ORGANIC ACID
BYPRODUCTS
a
b
[email protected])
Bioprocess and Biosystems Engineering (submitted)
401
Résumé
Avec l’absence d’émission de CO2 et une densité d'énergie massique plus élevée que
les carburants conventionnels, tels que l'essence, le diesel, le biodiesel et le bioéthanol,
le
biohydrogène est une option prometteuse en terme d'énergie renouvelable. Par
conséquent, la production biologique d'hydrogène, a une importance commerciale
lorsqu’il est produit à partir de matières premières provenant de déchets industriels à
faible coût. La fermentation sombre anaérobie est une méthode simple, efficace et
largement étudiée pour la production de biohydrogène. Au cours de la production
d'hydrogène par ce procédé, divers acides organiques sont accumulés diminuant ainsi
le pH du procédé. Par conséquent, pour éviter l'inhibition métabolique ou diminution de
rendement lors de la période de fermentation, un ajustement de pH rigoureux est
nécessaire. Pour la première fois, un système de bioréacteur anaérobie à deux phases
a été testé pour la production de biohydrogène et l'extraction in situ d'acides
organiques. Parmi les différents solvants testés, l'alcool oléique a été choisie en tant
que phase organique dans le procédé en raison de sa biocompatibilité. Un rapport de la
phase organique : aqueuse de 1: 50 a été déterminé comme étant optimal pour
augmenter la production d'hydrogène de 26.6 mmol/L à 29.6 mmol/L- milieu de culture.
Mots-clés: Biohydrogène; glycérol brut; alcool oléique; acide organique; bioréacteur en
deux phase
402
Abstract
Owing to CO2 free emission, hydrogen is considered as a potential green alternative of
fossil fuels. Water is the major emission of hydrogen combustion process and
gravimetric energy density hydrogen is nearly three times more than that of gasoline
and diesel fuel. Biological hydrogen production, therefore, has commercial significance;
especially, when it is produced from low cost industrial waste based feedstock. Light
independent anaerobic fermentation is simple and mostly studied method of
biohydrogen production. During hydrogen production by this method, a range of organic
acids are accumulated resulting in sharp decrease in process pH. Therefore, to avoid
process termination or possible metabolic shift, throughout the fermentation period a
rigorous pH adjustment is necessary. Alternatively, for the first time, a two phase
anaerobic bioreactor system has been reported for biohydrogen production and in situ
extraction of different organic acids. Among different solvents, based on biocompatibility
oleyl alcohol has been chosen as the organic phase of the process. An organic:
aqueous phase ratio of 1: 50 has been found to increase the hydrogen production from
26.58 mmol/L to 29.61 mmol/L-medium.
Keywords: Biohydrogen; crude glycerol; oleyl alcohol; organic acid; two phase
bioreactor
403
Introduction
Fossil fuels are extensively used to fulfill global energy demand. Unfortunately, it is a
limited resource and most importantly, over exploration of fossil fuel has adverse effect
on the environment (Ntaikou et al., 2010). In this context, a search for renewable and
sustainable energy source is going on throughout the world. As a result of this attempt,
biofuels such as biodiesel and bioethanol have become commercially available and
lately, biohydrogen is receiving much attention as a potential alternative of fossil fuels.
There are different reasons behind researchers’ the attention towards biohydrogen as a
renewable fuel. Firstly, almost all biomass, including different agro industrial wastes,
can be converted to hydrogen by microbial processes; although, hydrogen yield may
vary from feedstock to feedstock. Secondly, it is CO2 emission free fuel; major emission
of which is only water. Finally, gravimetric energy density of hydrogen is 142 MJ/kg;
which is approximatly three times of gasoline (47 MJ/kg) and diesel fuel (43 MJ/kg)
(Şensöz et al., 2000, Sydney et al., 2014).
Crude glycerol (CG) is a byproduct generated during trans-esterification of lipid used for
biodiesel production. For every 100 kg of biodiesel, nearly 10 kg of CG is produced
which may contain different compounds such as methanol, soap, monoglyceride,
diglyceride etc (Johnson et al., 2007). Mainly because of continuous global production
and crude nature, market value of CG could be as low as 0.05$/pound (Yang et al.,
2012). Therefore, researchers are looking for an economically sound method for CG
valorization. In this context, crude glycerol has been evaluated as a feedstock for
biohydrogen production by light independent anaerobic fermentation. During this
process along with hydrogen, a range of organic acids are produced as byproduct.
Accumulation of these acids results in sharp decrease in fermentation medium pH;
which may lead to termination of the process. Moreover, accumulation of organic acids
may initiate metabolic shift in an anaerobic process to produce more reduced end
products such as solvents (butanol) (Hawkes et al., 2002, Yusof et al., 2012), as a result
of which hydrogen production may decrease. Therefore, a novel two phase process has
been evaluated for potential application in hydrogen production. As shown in Figure
404
7.1.1, the organic phase of the process could be a biocompatible organic solvent
capable of extracting the organic acids produced during hydrogen production. The
aqueous phase will be CG based medium containing the microorganism. Thus, the
purpose of present investigation was to screen different organic solvent to identify the
most biocompatible one and to optimize process parameter such as volume of the
organic phase, for enhanced hydrogen production.
Microorganism and culture conditions
Enterobacter aerogenes NRRL B 407, a rod-shaped, gram-negative, facultative
anaerobic bacterium has been used for the present investigation. The bacterium, which
is known to produce hydrogen from a range of feedstock including waste biomass, was
provided by ARS (USDA, USA). The organism was routinely grown in a culture medium
composed of the following components (g/L): glucose (5), casein peptone (5), KH2PO4
(2), MgSO4 ̇ 7H2O (0.5) and yeast extract (0.5). For inoculum development, 50 mL of
aforementioned medium taken in serum bottle (125 mL) was bubbled with pure nitrogen
gas for 2 minutes, so that anaerobic condition can be created. After sterilization, the
bottles were inoculated and incubated using an incubator operated at 30 ±1° C and 150
rpm. For each experiment, 5% (v/v) of the medium containing freshly grown microbial
cell was used as inoculum (Sarma et al., 2013c).
Screening of different organic solvents for two phase system
Isopropyle ether ("Extraction of Acetic Acid from Water Using Isopropyl Ether" from the
Wolfram
Demonstrations
Project
http://demonstrations.wolfram.com/ExtractionOfAceticAcidFromWaterUsingIsopropylEth
er/ (accessed on 23/04/2014)), Tri-n-octyle amine (Rasrendra et al., 2011), Ethyle ether
(Kalaichelvi et al., 2007), Oleyl alcohol (Malinowski, 2001), 2-ethyle-1-hexanol
(Rasrendra et al., 2011) and Aliquat 336 (Katikaneni et al., 2002); a few solvents
commonly used for organic acid extraction, have been selected for present
investigation. This experiment was designed to evaluate the possibility of growing
Enterobacter aerogenes NRRL B 407 in presence of these solvents. Initial CG
405
concentration used for this investigation was 12 g/L. Sterile CG solutions were
distributed into different serum bottles in such a way that after inoculating with 5 % (v/v),
there would be 25 mL medium in each bottle. 2 mL of each solvent were filter sterilized
by 0.22 µ (25 mm) syringe filter (Fisher scientific, Canada) and separately added to
different serum bottles containing CG solutions. The two phase systems so obtained
were bubbled with nitrogen gas for 2 minutes to drive out the oxygen dissolved in the
liquid and the bottles were immediately closed with aluminum crimp seals and silicone
septa. By using a syringe fitted with hypodermic needle, 1.25 mL of inoculum prepared
using the procedure mentioned in section 2.1 was added to each bottle. Followed by
inoculation, the bottles were incubated for 96 hours in an incubator maintained at 30 ºC
and 150 rpm. In a similar manner, a serum bottle was prepared without any organic
solvent and used as control for comparison purpose. At the end of the experiment,
samples were collected from the aqueous phases of the systems and cumulative
biomass production and final pH was measured.
Hydrogen production with selected organic solvents
Based on the result of previous experiment, tri-n-octyle amine, oleyl alcohol and aliquat
336 were selected for further investigation. As mentioned in section 2.2; 23.75 mL of
CG solution was taken in serum bottle in such a way that after addition of 1.25 mL of
inoculum, total volume of the aqueous phase medium would become 25 mL and CG
concentration would be 12g/L. Different organic solvents were distributed in separate
serum bottles containing the CG solutions in such a way that for each organic solvent
three different organic: aqueous phase ratios, namely, 1: 25, 1: 12.5, and 1: 6.25, could
be obtained. One bottle was used as control where none of the solvents was added.
Liquids of each bottle was bubbled with nitrogen gas for 2 minutes and immediately
closed with crimp seal and septum as mentioned in section 2.2. All bottles were
sterilized in closed condition; cooled, inoculated with 1.25 mL of inocula and incubated
as described above. Using a gas tight syringe (SGE Analytical Science, Australia), from
the headspace of each bottle, 1 mL of gas sample was collected after 48 hours of
incubation and stored in gas tight vials until analyzed.
406
Investigation of the effect of oleyl alcohol and CG concentration on hydrogen
production
Based on the result of previous experiment (section 2.3), oleyl alcohol was selected to
be used in biohydrogen production process. It was observed that in the case of oleyl
alcohol, a relatively low organic: aqueous phase ratio was more favorable for hydrogen
production by Enterobacter aerogenes NRRL B 407. Therefore, hydrogen production at
different relatively low oleyl alcohol (organic phase): aqueous phase ratios, namely, 1:
100, 1: 50 and 1: 25 were compared with that of control medium with no organic phase.
Media preparation, sterilization, inoculation, incubation and sample collection conditions
were similar to the ones mentioned in section 2.3. Moreover, in an attempt to determine
the effect of CG concentration on hydrogen production by oleyl alcohol based novel two
phase system, another experiment was conducted using 25 g/L of CG. A set of serum
bottles containing only CG (25 g/L) were used as control whereas, in another set oleyl
alcohol was added at an organic: aqueous phase ratio of 1:25. Other conditions of the
experiments were similar to those mentioned above.
Analysis
Hydrogen analysis
In order to determine the hydrogen content, biogas samples which had been collected
during
the
experiments
were
analyzed
by
gas
chromatography.
The
gas
chromatography system used for this analysis was of Varian 3800 model (USA),
capable of headspace analysis. The column and detector used for this analysis were
PoraPLOT Q® (Agilent technology, USA) and thermal conductivity detector (TCD),
respectively. N2 at a flow rate of 3.5 mL/minute was used as the carrier gas whereas;
the column temperature was 100 °C.
Analysis of fermentation end products
In order to analyze the fermentation end products, at the end of each experiment liquid
samples were collected from the aqueous phase of the system. They were centrifuged
for 5 minutes at 10000 rpm; supernatants were filtered by 0.45 µ syringe filter and
407
analyzed by GC. The GC (GC7890B, Agilent Technologies, USA) used for this analysis
was fitted with flame ionization detector (FID). The column temperature gradient used
for this analysis was 50-250 ºC. It was achieved by increasing the temperature at a rate
of 20 ºC per minute. Injection volume was 0.8 µl and isobutanol was used as internal
standard for all samples.
Screening of different organic solvents for two phase system
Biocompatibility of the organic phase is highly desirable for a two phase reactor system
used for bioconversion purpose. Therefore, six different solvents considered for the
present investigation were tested for possible inhibitory effect on biomass production of
Enterobacter aerogenes NRRL B 407, the hydrogen producing bacterium used in this
study. The results of this investigation have been presented in Figure 7.1.2. From
Figure 7.1.2 it is evident that, in the case of oleyl alcohol biomass production was found
to be nearly 60 mg/L; which is 50 % more than the control experiment conducted in the
absence of any solvent. However, presence of other solvents was found to have
adverse effect on biomass production. Among rest of the solvents biomass production
was slightly higher in the case of tri-n-octyle amine and aliquat 336; hence, along with
oleyl alcohol, they were also considered for further investigation. At the end of the
process, aqueous phase pH of the two phase systems corresponding to different
solvents was measured. As shown in Figure 7.1.2, compared to pH 4.65 found for
control, for oleyl alcohol the same was found to be 5.15. This observation indicates that
oleyl alcohol was capable of extracting the organic acids accumulated in the aqueous
phase to resist sharp decrease in media pH; which is, otherwise, commonly observed in
the case of batch hydrogen production processes. From Figure 7.1.2 it is also observed
that final media pHs of two phase systems involving tri-n-octyle amine, ethyle ether, 2ethyle-1-hexanol, and isopropyle ether were also higher than single phase control.
However, in those cases microbial growth was negligible; which indicates that only a
small amount of organic acids were there in the aqueous phase of those systems.
408
Hydrogen production with selected organic solvents
The purpose of this investigation was to determine the effect of organic phase volume
on hydrogen production. As mentioned in previous section, oleyl alcohol, tri-n-octyle
amine and aliquat 336 were chosen for this investigation and organic phase: aqueous
phase ratios considered were 1: 25, 1: 12.5, and 1: 6.25. The results of this
investigation have been presented in Figure 7.1.3. From Figure 7.1.3 it is clear that at all
organic phase: aqueous phase ratios, tri-n-octyle amine and aliquat 336 were inhibitory
for hydrogen production and hence they were not considered for further investigation.
On the contrary, in the case of oleyl alcohol, corresponding to decrease in organic:
aqueous phase ratio from 1: 6.25 to 1.25; gradual improvement in hydrogen production
has been observed (Figure 7.1.3). Probably, a thick organic layer on top of the aqueous
phase medium posed as a barrier for hydrogen to release into the headspace of the
reactor. Additionally, a large volume of the organic phase may interfere in N2 bubbling
process, preventing sufficient removal of oxygen from the media.
Based on this
observation it has been postulated that by further decreasing the organic phase volume,
improved hydrogen production could be possible. Thus, as explained in next section
(3.3) another experiment was carried out using oleyl alcohol with yet lower organic
phase volume.
Investigation of the effect of oleyl alcohol volume and CG concentration on
hydrogen production using proposed two phase system
As a part of this investigation, the effect of oleyl alcohol phase volume on hydrogen
production by E. aerogenes has been investigated and the results have been presented
in Figure 7.1.4. Figure 7.1.4 shows that application of an organic phase comprising of
oleyl alcohol has beneficial effect on hydrogen production; which can be improved by
reducing the organic: aqueous phase ratio from 1:25 to 1: 50. By using a phase ratio of
1: 50, hydrogen production can be increased from 26.58mmol/L to 29.61mmol/Lmedium. However, further increase in the ratio to 1: 100 did not show any significant
enhancement in hydrogen production. Probably, at this ratio, organic acid extraction by
the organic phase was not sufficient to make any significant impact on hydrogen
409
production process. As already mentioned in section 2.5.2, aqueous samples were
analyzed by GC to determine the fermentation end products. In Figure 7.1.5,
fermentation end product profiles of control and two phase system involving oleyl
alcohol at a phase ratio of 1: 50 have been presented. From Figure 7.1.5 it is evident
that, the butyric acid peak seen at 8.45 min of the control sample became
unrecognizable due to presence of oleyl alcohol phase. Likewise, the peak at 5.48 min,
most probably for a low molecular weight organic acid such as formic acid, also
disappeared in the aqueous sample collected from the proposed two phase reactor.
This observation indicates that the organic phase was capable of extracting the organic
acids from the aqueous phase of the reactor and it could be related to observed
increase in hydrogen production.
CG concentration is also a crucial factor in hydrogen production by CG bioconversion. It
has been observed that for a batch process, 10 g/L CG is optimum for hydrogen
production (Sarma et al., 2013b). Increase in CG concentration beyond this
concentration resulted in almost no increase in hydrogen production. Moreover,
corresponding to an increase in CG concentration from 2.5 g/L to 20 g/L, gradual
decrease in final media pH was observed (Sarma et al., 2013b). It suggests that at
relatively high initial CG concentration, more amounts of organic acids accumulate in
the medium. Thus, simultaneous extraction of these organic acids might have beneficial
effect on cumulative hydrogen production. Therefore, proposed two phase system was
tested for hydrogen production; where, initial CG concentration was as high as 25 g/L.
Results of this investigation has been presented in Figure 7.1.6. From Figure 7.1.6 it is
evident that in contrast to 1.48 mmol hydrogen produced per liter medium of a single
phase process; it was as high as 11.65 mmol per liter medium of proposed two phase
system. High glycerol concentration is known to have inhibitory effect on hydrogen
production (Ito et al., 2005); therefore, for further improvement in product yield substrate
inhibition should be avoided by using a continuous process. Overall, the observation
suggests that simultaneous extraction of organic acid accumulated in the medium has
positive effect on hydrogen production. Therefore, the concept deserves further
exploration. For instance, instead of carrying out fermentation and extraction in the
410
same vessel; a dedicated extraction vessel can be introduced for continuous extraction
of organic acids and extracted medium can be recirculated to the fermentation vessel
for further fermentation. Additionally, if the organic acid byproducts are extracted,
without further sterilization remaining water can be reused. It may save both energy and
water required for hydrogen production. For bioconversion of 1 kg of CG as high as 400
L of water is required (Sarma et al., 2013a); therefore, reutilization of water is inevitable
for large scale hydrogen production. However, direct application of large volume (more
than 10 % v/v) of organic acid rich wastewater from preceding batch was found to be
inhibitory for hydrogen production (Sarma et al., 2013b). Thus, further investigation on
simultaneous extraction of organic acid will be beneficial for fermentative hydrogen
production.
Conclusions
During hydrogen production by light independent anaerobic fermentation, organic acid
byproducts are accumulated in the medium; which is detrimental for the process. To get
rid of the problem, a novel two phase process for hydrogen production and
simultaneous extraction of organic acid has been proposed. Owing to its
biocompatibility, oleyl alcohol has been chosen for in situ extraction of organic acids. An
organic: aqueous phase ratio of 1: 50 was found to be optimum for the process. For a
process with initial CG concentration of 25 g/L, compared to 1.48 mmol-H2/L-medium of
a single phase process; as high as 11.65 mmol-H2/L-medium has been obtained by
proposed two phase system. At high initial substrate (CG) concentration such as 25 g/L,
organic acid concentration in the medium is more; therefore, the approach should be
more appropriate for such process. Moreover, the strategy has the potential to reduce
the volume of water required for large scale hydrogen production.
Acknowledgments
411
References
Hawkes FR, Dinsdale R, Hawkes DL & Hussy I (2002) Sustainable fermentative
hydrogen production: challenges for process optimisation. International Journal
of Hydrogen Energy 27(11–12):1339-1347.
production
from
glycerol-containing
wastes
discharged
after
biodiesel
Johnson DT & Taconi KA (2007) The glycerin glut: Options for the value-added
conversion of crude glycerol resulting from biodiesel production. Environmental
Progress 26(4):338-348.
Kalaichelvi P, Perumalsamy M, Arunagiri A & Sofiya K (2007) Synergistic extraction of
acetic acid from its aqueous solution. Journal of the University of Chemical
Technology and Metallurgy 42(3):291-294.
Katikaneni SP & Cheryan M (2002) Purification of fermentation-derived acetic acid by
liquid-liquid extraction and esterification. Industrial & Engineering Chemistry
Research 41(11):2745-2752.
Malinowski JJ (2001) Two-phase partitioning bioreactors in fermentation technology.
Biotechnology advances 19(7):525-538.
Ntaikou I, Antonopoulou G & Lyberatos G (2010) Biohydrogen production from biomass
and wastes via dark fermentation: a review. Waste and Biomass Valorization
1(1):21-39.
Rasrendra C, Girisuta B, Van de Bovenkamp H, Winkelman J, Leijenhorst E,
Venderbosch R, Windt M, Meier D & Heeres H (2011) Recovery of acetic acid
from an aqueous pyrolysis oil phase by reactive extraction using tri-n-octylamine.
Chemical Engineering Journal 176:244-252.
412
Sarma SJ, Brar SK, Le Bihan Y, & Buelna G, Bio-hydrogen production by biodieselderived crude glycerol bioconversion: a techno-economic evaluation, Bioprocess
and Biosystems Engineering, 36 (2013a) 1-10.
Sarma SJ, Dhillon GS, Brar SK, Le Bihan Y, Buelna G & Verma M (2013c) Investigation
of the effect of different crude glycerol components on hydrogen production by
Enterobacter aerogenes NRRL B-407. Renewable Energy 60:566-571.
Şensöz S, Angın D & Yorgun S (2000) Influence of particle size on the pyrolysis of
rapeseed (Brassica napus L.): fuel properties of bio-oil. Biomass and Bioenergy
19(4):271-279.
nutrient source. Bioresource Technology 159:380-386.
Yang F, Hanna M & Sun R (2012) Value-added uses for crude glycerol--a byproduct of
biodiesel production. Biotechnology for Biofuels 5(1):13.
Yusof SJHM, Takriff MS, Kadhum AAH, Mohammad AW & Jahim J (2012) The effect of
initial butyric acid addition on ABE fermentation by C. acetobutylicum NCIMB
619. International Proceedings of Chemical, Biological & Environmental
Engineering 48.
413
Figure 7.1. 1: Schematic representation of the proposed method
414
6.5
Biomass
60
pH
6
50
5.5
40
5
30
4.5
20
4
10
3.5
0
3
Figure 7.1. 2: Screening of organic phase based on biocompatibility & final media pH
415
pH
Biomass production (mg/L)
70
30
25
20
15
10
5
0
Figure 7.1. 3: Screening of organic phases based on hydrogen production
416
31
30
29
28
27
26
25
24
Oleyl alcohol
(1:100)
Oleyl alcohol
(1:50)
Oleyl alcohol
(1:25)
Control
Figure 7.1. 4: Investigation of the effect of oleyl alcohol: aqueous phase ratio on hydrogen
production
417
Figure 7.1. 5: Fermentation end products detected in the aqueous phase of the reactor. (a) Single
phase control, (b) two phase process containing oleyl alcohol at an organic: aqueous phase ratio
of 1: 50.
418
14
12
10
8
6
4
2
0
Oleyl alcohol (1:25)
Control (CG 25g/L)
Figure 7.1. 6: Investigation of the effect of high CG concentration on hydrogen production using
novel two phase system
419
PART II
LOW COST SEMI-CONTINUOUS BIOPROCESS AND ONLINE
MONITORING OF HYDROGEN PRODUCTION FROM CRUDE
GLYCEROL
Mausam Vermac
a
b
Centre de Recherche Industrielle du Québec (CRIQ), Québec (QC), G1P 4C7, Canada
c
CO2 Solutions Inc., 2300, rue Jean-Perrin, Québec, Québec G2C 1T9 Canada
[email protected])
Cet article a dû être retiré de la version électronique en raison de restrictions liées au
droit d’auteur.
Environmental Science and Technology (submitted, possibility of US patent)
421
CHAPITRE 8
CONCLUSIONS ET RECOMMANDATIONS
Chapitre 8. Conclusions at Recommandations
Conclusions
Les principales conclusions de la thèse de doctorat sont présentées ci-dessous:
1. Les rejets de l’industrie de biodiesel, particulièrement riche en glycérol,
représentent un milieu favorable pour la production d'hydrogène par voie
microbienne. Dans les travaux réalisés dans cette thèse, 10 g / L de glycérol brut
(GB) s’est avérée la concentration optimale pour la production de H2.
2. Le Ni, le Fe, le Mo et le S sont les éléments essentiels pour la synthèse des
enzymes (hydrogénases et nitrogénases) intervenant dans la production de
l’hydrogène. Un milieu équilibré en ces éléments favorise et améliore la production
d'hydrogène.
3. L’éthanol, le 1,3 propanediol, l'acide butyrique et le méthane sont les principaux
sous-produits à grande importance commerciale générés durant la production
d'hydrogène par voie fermentaire.
4. Le coût des différents suppléments ajoutés au GB pour la production d'hydrogène tel
que l’addition de rejets solides de l’industrie de bière (50 mg / L) comme source
d'éléments nutritifs permet d’améliorer la production de H2 de 32,50% avec un taux
maximum de production journalier de 1040 mL/L. Il a été également observé que
l'addition des déchets liquides d’abattoir (SL), de la biomasse des déchets de
brasserie (BWB) et de l'urée au GB peut améliorer la production d'hydrogène de
respectivement 18,81 ± 3,56; 27,30 ± 3,54 et 38,57 ± 3,66%.
5. En utilisant la méthodologie de surface de réponse, l’effet du savon, du méthanol et
du NaCl sur la production d'hydrogène et la bioconversion du glycérol a été évalué.
le savon (1 à 3 g/L) avait un effet inhibiteur significatif (p = 0,0104 ˂ 0,05) sur la
production d'hydrogène. De même, le méthanol (1.3 à 3.3 g / L) avait un effet
similaire dans la plage de concentration étudiée.
6. La désalaison peut éliminer jusqu’à environ 42% de savon présent dans le GB.
Cependant, son élimination affecte négativement la production de H2 (le savon est
utilisé comme co-substrat pour la production de H2). le traitement du GB par du
MgSO4 permet de convertir le savon en sa forme inactive (écume). Cette nouvelle
447
approche permet d’augmenter la production de H2 de 34,70% et la bioconversion du
glycérol de 2,5 fois. L’utilisation de MgSO4 permet également d’améliorer la qualité
nutritionnelle du GB vu qu’il contient seulement 57 ± 18 mg/L.
7. Des nanoparticules de citrate de fer, de FeSO4, NiCl2 et de l'acétate de nickel ont
été préparées en utilisant la technologie de séchage par nano-pulvérisation. L’effet
de ces nanoparticules sur la production de H2 a été étudié. Seules les particules de
citrate ferrique avaient un effet bénéfique sur la production de H2. Deux types de
nanoparticules de citrate de fer ont été préparés, par dissolution dans de l'eau
chaude, et dissolution dans une solution de NaOH. Les nanoparticules préparées
par dissolution dans de l'eau chaude se sont avérées capables d’améliorer la
production d'hydrogène de 50, 5%.
8. La concentration initiale en GB est le facteur principal qui affecte le rendement en
hydrogène. À des concentrations initiales extrêmement faibles (de l’ordre de 100
mg/L) et en utilisant les particules de citrate de fer, le maximum du rendement en
hydrogène atteint était de 29,9 mol H2/kg GB. Ce rendement est nettement
supérieur à 8,25 mol H2/kg GB, valeur obtenue dans le cas de fermentation obscure.
9. Les rejets liquides issus de la production du biohydrogène (HPLW) ont d’excellentes
propriétés de solubilisation du phosphate. Ces rejets ont
un fort potentiel
commercial puisqu’ils peuvent être utilisés comme une alternative aux bioengrais.
Avec ces rejets, on peut augmenter la concentration en phosphate soluble 23,17 fois
en 1 minute d'application. Contrairement aux bioengrais traditionnelles, ces rejets
ne renferment pas de microorganismes vivants et par conséquent, la durée de
conservation est plus longue pour ces produits (rejets) proposés.
10. Des plantes de soya cultivés dans du sol enrichi avec du HPLW ont présenté une
capacité d’absorption du phosphore 2,18 à 2,74 fois supérieure par rapport au
contrôle (plantes cultivées dans du sol sans HPLW). Grâce à ces résultats, les
HPLW peuvent être présentés comme étant un produit à fort pouvoir commercial et
une alternative intéressante aux bioengrais phosphatés actuellement sur le marché.
11. Durant la production du biohydrogène, l'accumulation des acides organiques (sousproduits de cette fermentation obscure et anaérobie) entraîne une diminution
448
considérable du pH du milieu ce qui est critique pour le procédé. Pour remédier à
cette problématique, un nouveau procédé biphasique a été développé, permettant la
production d'hydrogène et, en même temps, l'extraction de l'acide organique généré.
En raison de sa biocompatibilité, l'alcool oléique a été sélectionné pour l'extraction
in situ de ces acides organiques. Un rapport phase organique/phase aqueuse de 1:
50 était l’optimal pour ce procédé, ce qui permet d'augmenter la production
d'hydrogène de 26,58 mmol / L à 29,61 mmol / L.
12. L'acétone, l'éthanol et le butanol ont été détectés dans le GB (substrat) utilisé dans
la présente étude. L'acétone-butanol-éthanol (ABE) sont convertis en GB par action
de la microflore indigène durant le stockage.
13. En le diluant avec de l'eau, le GB peut être utilisé seul, sans aucun ajout de
suppléments pour la conception d’un procédé de production de H2 en mode semicontinu et à faible coût, avec une production améliorée d'hydrogène. Avec ce
procédé, il est possible d’atteindre 5,18 L-H2 /L, ce qui est équivaut à 210 mmol-H2
/L. Cette productivité est nettement plus élevée que 2,02 à 2,68 L H2 / L, valeur
moyenne de productivité d'hydrogène à partir du GB.
449
Recommandations pour l'avenir
Sur la base des résultats issus de cette thèse de doctorat, les recommandations
suivantes peuvent être proposées pour la continuité de la recherche.
1. Pour parvenir à une production optimale en biohydrogène à partir du glycérol brut
(GB) et, à partir de n’importe quel autre substrat d’une façon générale, les procédés
de fermentation devraient assurer le maximum de bioconversion de cette matière
première. Par exemple, les procédés fonctionnant en mode "fed-batch " et en
mode "en continu " devront être considérés.
2. Une grande quantité d'eau est utilisée durant la production de biohydrogène, cette
quantité pourrait atteindre jusqu’à 400 L/kg de substrat. Pour une production
durable d’hydrogène, cette eau devrait être réutilisée.
3. D’autres études supplémentaires devront être réalisées afin d’exploiter au
maximum les avantages du procédé biphasique proposé dans cette thèse. Au lieu
d’utiliser un seul compartiment
au sein du bioréacteur pour les deux phases
aqueuse et organique, l’étape d'extraction peut être effectuée dans un deuxième
compartiment dédié pour ce fait. En utilisant cette approche, Il est possible de
contourner les problèmes dûs à la présence de la phase organique durant
l’extraction d’biohydrogène.
4. Pour atteindre un taux de production d'hydrogène élevé ainsi qu’une productivité
améliorée, le processus devrait être étudié à haute densité initiale de cellules.
5. Afin de réduire la quantité d'énergie nécessaire pour la production biologique
d'hydrogène, la stérilisation classique à la chaleur peut être remplacée par de
nouvelles
technologies
de
stérilisation. :
la
destruction
sélective
des
microorganismes indésirables par traitement chimique peut être une technologie
intéressante.
6. Il sera intéressant si toute l'énergie nécessaire à la production d'hydrogène
biologique pourrait être obtenue à partir d'une source d'énergie renouvelable telle
que l'énergie solaire.
450
ANNEXES
Annexes
ANNEXE I
Data: Hydrogen biorefinery: Potential utilization of the liquid waste from
fermentative hydrogen production
Figure 2.3.2: Reported concentration levels of different metabolites present in HPLW and their market
price in US dollars (Han et al., 2010, Lalaurette et al., 2009, Selembo et al., 2009).
1,3propandiol
Ethanol
Acetic
Butyric
Succnic
acid
acid
acid
Lactic acid
Propionic
Valeri
acid
c acid
yield
(mg/L)
1710.28
895.69
764.64
168.86
660
160
18.6
14.72
288
134.6
55.25
74.6
226
78
45.5
37.4
Price
($/L)
452
Annexes
Figure 2.3.3: Free energy of formation (25º C) of different metabolites produced during biohydrogen
production.
Methane
-50.79
Ethanol
-181.75
1,3-propandiol
-326.93
Butyric acid
-377.8
Acetic acid
-396
Propionic acid
-383.5
Lactic acid
-540
Succnic acid
-746.38
valerate
-344.34
453
Annexes
ANNEXE II
Data:
Bio-hydrogen
production
by
biodiesel
derived
crude
bioconversion: a techno-economic evaluation
Figure 2.4.2: Cost distribution of the crude glycerol (1 kg) bioconversion process
Item/process
Cost ($)
CG pretreatment
2.38
Dark fermentation (inoculum media cost)
44.88
Dark fermentation (inoculum electricity cost)
0.037
Dark fermentation (media cost)
161.04
Dark fermentation (electricity cost)
2.14
Photo-fermentation (inoculum media cost)
44.88
Photo-fermentation (inoculum electricity cost)
0.037
Photo-fermentation (media cost)
25.88
Photo-fermentation (electricity cost)
21.83
Equipment
29.98
CG
0.33
Total Cost
333.414
454
glycerol
Annexes
Figure 2.4.3: Electricity requirement of a two-stage fermentation process for CG (1 kg) bioconversion and
H2 production
Process
Electricity required (kWh)
Dark fermentation
48.85
Photo-fermentation
490.45
455
Annexes
ANNEXE III
Data: Hydrogen production from meat processing and restaurant waste derived crude glycerol by anaerobic
fermentation and utilization of the spent broth
Figure 3.1.1: Batch H2 production and glycerol utilization profiles obtained for different initial CG concentrations
Time
Hydrogen production (mmol)
Residual glycerol concentration (%)
(h)
2.5 g/L CG
5 g/L CG
10 g/L CG
15 g/L CG
20 g/L CG
2.5 g/L GC
5 g/L CG
10 g/L CG
15 g/L CG
20g/L CG
0
0
0
0
0
0
100
100
100
100
12
0.79
0.49
0.83
0.45
0.74
40.50
83.93
77.87
78.25
98.24
24
1.21
0.95
1.45
0.87
1.32
7.84
57.2
71.25
69.10
88.26
36
1.21
1.66
2.11
1.57
1.91
0.17
36.4
54.88
63.82
79.93
48
1.21
1.99
2.67
2.03
2.32
0.17
19.8
49.28
58.94
69.82
60
1.21
2.28
3.19
2.53
2.86
0.17
10.73
36.76
56.30
65.2
72
1.21
2.32
3.45
2.82
3.24
0.17
5.32
30.60
50.60
63.59
456
100
Annexes
84
1.21
2.35
3.71
3.20
3.48
0.17
2.34
23.92
40.85
58.43
96
1.21
2.35
3.83
3.45
3.73
0.17
2.55
19.17
33.33
52.80
120
1.21
2.35
4.00
3.86
3.98
0.17
0.77
13.38
29.47
44.51
144
1.21
2.35
4.12
4.07
4.19
0.17
1.11
8.24
29.06
41.15
168
1.21
2.35
4.2
4.24
4.32
0.17
1.74
5.92
28.04
40.95
457
Annexes
Figure 3.1.2: pH profiles obtained from the batch experiments conducted for different (initial) CG
concentrations
Time (h)
2.5 g/L CG
5 g/L CG
10 g/L CG
15 g/L CG
20 g/L CG
0
4.43
4.53
4.61
4.64
4.67
12
3.9
4
3.92
4
3.97
24
3.74
3.87
3.81
3.83
3.81
36
3.84
3.79
3.83
3.75
3.71
48
3.86
3.75
3.73
3.69
3.65
60
3.92
3.78
3.73
3.72
3.65
72
3.93
3.73
3.7
3.67
3.6
84
4.06
3.83
3.75
3.71
3.62
96
3.98
3.86
3.77
3.72
3.61
120
4
3.88
3.8
3.75
3.65
144
4.1
3.89
3.84
3.73
3.67
168
4.06
3.9
3.81
3.69
3.65
458
Annexes
Figure 3.1.6: Batch H2 production profiles obtained by addition of different concentrations of spent
biomass (SB) (autoclaved) from previous fermentation
Time (h)
SB 5 mg/L
SB 10 mg/L
SB 50 mg/L
SB 150 mg/L
0
0
0
0
0
12
1.16
1.16
1.28
1.16
24
2.12
2.15
2.28
2.16
36
2.61
2.65
2.82
2.78
48
3.28
3.23
3.53
3.49
60
3.49
3.44
3.94
3.86
72
3.74
3.69
4.40
4.28
84
3.90
3.86
4.78
4.57
96
3.99
3.94
4.98
4.78
120
4.15
4.02
5.27
5.03
144
4.24
4.07
5.48
5.19
168
4.24
4.07
5.57
5.28
459
Annexes
0.8
y = 0.0404x - 0.0081
R² = 0.9987
Absorbance (410 nm)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
-0.1
0
5
10
glycerol (mg/L)
15
Figure: Standard curve for determination of glycerol concentration
460
20
Annexes
ANNEXE IV
Data: Evaluation of different supplementary nutrients for enhanced biohydrogen
glycerol
Figure 3.2.2: Maximum cumulative H2 production (mmol/L), glycerol utilization (%) and broth pH recorded
at the end of fermentation for different material supplemented to 10 g/L CG. The control indicates the
results obtained for 10 g/L CG without any additive.
Response
Urea 10
BWB 10
SL 10
SS 20
BWL 20
AP 10
SIW 1%
CG 10g/L
mg/L
mg/L
mg/L
mg/L
mg/L
mg/L
(v/v)
(control)
H2 production
(mmol/L)
116.4
106.92
99.8
94.00
92.2
72.4
47.4
84
Final pH
3.3
3.3
3.38
3.26
3.26
3.41
3.57
3.81
93.40
84.50
93.60
87
90.5
72.135
61.00
94.08
Glycerol
utilization (%)
461
Annexes
Figure 3.2.3: H2 production profiles obtained for different concentrations urea supplemented to 10 g/L CG
Time
(h)
10 mg/L
20 mg/L
60 mg/L
120 mg/L
180 mg/L
0
0
0
0
0
0
12
1.18
0.16
0.12
0
0
24
2.26
0.91
1.08
0.9
0
36
3.18
2.83
2.40
1.9
0
48
4.01
3.16
3.15
2.37
0
60
4.63
3.20
3.40
2.62
0
72
5.05
3.24
3.40
2.62
0.04
84
5.34
3.28
3.40
2.62
0.45
96
5.50
3.33
3.40
2.62
0.70
120
5.7
3.33
3.49
2.62
0.95
144
5.82
3.33
3.49
2.62
1.12
168
5.82
3.33
3.49
2.62
1.12
462
Annexes
ANNEXE V
Data: Mitigation of the inhibitory effect of soap by magnesium salt treatment of
crude glycerol- a novel approach for enhanced biohydrogen production from the
biodiesel industry waste
Figure 4.2.1: Cumulative H2 production (after 120h) obtained using 10 g/L CG (treated with different
concentration of NaCl). Initial NaCl concentration in the medium of each run of experiment was also
shown
Response
1g/L
5g/L
10g/L
20g/L
50g/L
100g/L
200g/L
Control
Cumulative H2
production: mmol/L
30.49
38.38
39.72
39.00
21.23
14.58
2.32
33.3
0.05
0.25
0.5
1.00
2.5
5.00
10
0
NaCl (g/L) in final
medium
463
Annexes
Figure 4.2.3: H2 production (a) and glycerol utilization (b) profiles obtained for 20 g/L CG treated with
different concentrations of MgSO4. Control represents result obtained for untreated CG
(a) Cumulative H2 production (mmol/L)
MgSO4 (0.5g/L)
Time (h)
MgSO4 (1g/L)
MgSO4
MgSO4
Control (CG 20
(2.5g/L)
(5g/L)
g/L)
0
0.99
0.99
0.99
0.99
0.92
12
6.50
6.06
7.63
3.69
5.07
24
11.98
16.09
13.37
4.55
12.70
36
14.9
21.7
16.37
4.13
16.74
48
14.71
23.83
16.62
4.25
17.7
(b) Residual glycerol (%)
Time (h)
MgSO4 (0.5 g/L)
MgSO4 (1 g/L)
MgSO4 (2.5
MgSO4 (5
Control (CG20
g/L)
g/L)
g/L)
0
100
100
100
100
100
24
93.96
88.93
89.83
96.74
96.15
48
87.06
79.05
87.71
97.24
91.34
464
Annexes
25000
y = 46.257x + 402.82
R² = 0.9963
GC peak area
20000
15000
10000
5000
0
0
100
200
300
400
Hydrogen in the sample (µl)
500
Figure: Standard curve for determination of hydrogen concentration
465
600
Annexes
ANNEXE VI
Data: Enriched hydrogen production by bioconversion of biodiesel waste
supplemented with ferric citrate and its nano-spray dried particles
Figure 5.1.4: Cumulative hydrogen production obtained after 48 h of incubation. Different concentrations
of two different types of ferric citrate particles were added to 12 g/L of CG
Particle type
10 mg/L
50 mg/L
100 mg/L
300 mg/L
500 mg/L
13.03
9.6
10.34
6.95
6.54
26.58
33.64
36.49
37.37
40.00
NaOH based
particles
Water based
particles
466
Annexes
Figure 5.1.5: Cumulative hydrogen production obtained after 48 h of incubation. 100 mg/L of ferric
citrated particles (prepared with hot water) were added to different concentrations of CG. Projected
hydrogen yields by bioconversion of 1 kg of CG have also been shown for each initial CG concentration
Response
100
500
mg/L
mg/L
1 g/L
2 g/L
5 g/L
10 g/L
Cumulative H2 production (mmol/L)
3.00
5.97
11.23
18.24
32.53
36.45
Cumulative H2 production (mol/kg)
29.9
11.94
11.23
9.12
6.50
3.64
467
Annexes
ANNEXE VII
Data: Liquid waste from bio-hydrogen production - a commercially attractive
alternative for phosphate solubilizing bio-fertilizer
Figure 6.1.2: Phosphate solubilization achieved using different concentrations of bio-hydrogen production
liquid waste. The control represents soluble phosphate concentration obtained using only water as the
solubilizing agent
Response
Bio-hydrogen production liquid waste
Control 0%
(v/v)
Soluble
10% (v/v)
20% (v/v)
40% (v/v)
60% (v/v)
100% (v/v)
1.38
4.83
8.65
15.71
20.50
32.06
2.7
3.23
5.84
25.73
35.78
49.98
phosphate
(mg/L)
468
Annexes
3.5
y = 0.6259x + 0.0384
R² = 0.9997
Absorbance (827nm)
3
2.5
2
1.5
1
0.5
0
0
1
2
3
4
Soluble phosphate (mg/L)
5
6
Figure: Standard curve used to determine the soluble phosphate concentration
469
Annexes
ANNEXE VIII
Data: Phosphate solubilization using biohydrogen production liquid waste and
improved phosphorus uptake by soybean plant
Figure 6.2.3: Phosphorus uptake by soybean plants cultivated in the presence of different amounts of
hydrogen production liquid waste (HPLW) and tricalcium phosphate (TCP).
50 mL
Response
100mL HPLW
50mL HPLW
Soil control
HPLW+Soil+TCP
Soil+TCP
38.46
30.58
14.01
43.35
39.81
Phosphate (µg/g
biomass)
470
Annexes
Figure 6.2.4: Effect of hydrogen production liquid waste (HPLW) and tricalcium phosphate (TCP)
treatment of soil on soybean plant biomass production and seed germination.
Response
100mL HPLW
50mL HPLW
Soil control
50 mL HPLW+Soil+TCP
Soil+TCP
of 10 plants
3.66
6.04
10.27
8.35
9.19
Germination (%)
12.5
25
18.75
37.5
12.5
Total biomass (g)
471
Annexes
ANNEXE IX
Data: A novel anaerobic two phase system for biohydrogen production and in situ extraction organic acid
byproducts
Figure 7.1.2: Screening of organic phase based on biocompatibility & final media pH
2-ethyle-1Organic phase (2 mL)
Isopropyle ether
Tri-n-octyle amine
Ethyle ether
Oleyl alcohol
hexanol
Aliquat 336
Control
(mg/L)
6.66
20.00
13.33
60.00
6.66
26.66
40
pH
5.82
6.03
5.74
5.15
6.13
3.18
4.65
Biomass production
472
Annexes
Figure 7.2.3: Screening of organic phases based on hydrogen production
Organic
Oleyl
Oleyl
Oleyl
Aliquat
Aliquat
Aliquat 336
Tri-n-octyle
Tri-n-octyle
Tri-n-octyle
phase:
alcohol
alcohol
alcohol
336 (1:25)
336
(1:6.25)
amine (1:25)
amine (1:12.5)
amine
medium
(1:25)
(1:12.5)
(1:6.25)
(1:12.5)
Control
(1:6.25)
H2
production
(mmol/L)
27.50
23.64
8.78
1.22
0.97
473
0.80
0.73
0.64
0.91
26.58

production d`hydrogène par bioconversion du

Transcription

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